Protein tyrosine phosphatase-inhibiting compounds

ABSTRACT

Y—X—C(R′)═C(R″)COOR′″  (A1) 
       
     
     The present invention relates to novel protein tyrosine phosphatase modulating compounds having the general structure shown in Formula (A1), to methods for their preparation, to compositions comprising the compounds, to their use for treatment of human and animal disorders, to their use for purification of proteins or glycoproteins, and to their use in diagnosis. The invention relates to modulation of the activity of molecules with phosphotyrosine recognition units, including protein tyrosine phosphatases (PTPases) and proteins with Src-homology-2 domains, in in vitro systems, microorganisms, eukaryoic cells, whole animals and human beings. R′ and R″ are independently selected from the group consisting of hydrogen, halo, cyano, nitro, trihalomethyl, alkyl, arylalkyl. R′″ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, arylalkyl, X is aryl, Y is selected from hydrogen or                    
     wherein (*) indicates a potential point of attachment to X.

This application is a divisional application of U.S. patent applicationSer. No. 08/543,630, filed Oct. 19, 1995, now issued as U.S. Pat. No.5,770,620; which claims priority under 35 U.S.C. §119(e) of U.S.Provisioal Application Ser. No. 60/017,610, filed Jun. 19, 1995.

FIELD OF THE INVENTION

The present invention relates to novel protein tyrosine phosphatasemodulating compounds, to methods for their preparation, to compositionscomprising the compounds, to their use for treatment of human and animaldisorders, to their use for purification of proteins or glycoproteins,and to their use in diagnosis. The invention relates to modulation ofthe activity of molecules with phosphotyrosine recognition units,including protein tyrosine phosphatases (PTPases) and proteins withSrc-homology-2 domains, in in vitro systems, microorganisms, eukaryoiccells, whole animals and human beings.

BACKGROUND OF THE INVENTION

Reversible phosphorylation of proteins is a prevalent biologicalmechanism for modulation of enzymatic activity in living organisms.Tonks et al., J. Biol. Chem., 263(14):6722-30 (1988). Such reversiblephosphorylation requires both a protein kinase (PK), to phosphorylate aprotein at a particular amino acid residue, and a protein phosphatase(PP), to remove the phosphate moieties. See generally, Hunter, Cell,80:225-236 (1995). Recently, it has been estimated that humans have asmany as 2000 conventional PK genes, and as many as 1000 PP genes. Id.

One major class of PK's/PP's—the protein seine/threonine kinases andprotein serine/threonine phosphatases—have been shown to play criticalroles in the regulation of metabolism. See generally, Cohen, TrendsBiochem. Sci., 17:408-413 (1992); Shenolikar, Ann. Rev. Cell Biol.,10:55-86 (1994); Bollen et al., Crit. Rev. Biochem. Mol. Biol.,27:227-81 (1992). As their name suggests, these enzymes phosphorylateand dephoshphorylale serine or threonine residues of substrate proteins.Inhibitors of prntein serine/threonine phosphatases and kinases havebeen described. See, e.g., MacKintosh and MacKintosh, TIBS, 19:444-448(1994).

The protein tyrosine kinases/phosphatases comprise a second, distinctfamily of PK/PP enzymes of significant interest, and have beenimplicated in the control of normal and neoplastic cell growth andproliferation. See Fisher et al., Science, 253:401-406 (1991) Proteintyrosine kinase (PTK) genes are ancient in evolutionary origin and sharea high degree of inter-species conservation. See generally Hunter andCooper, Ann. Rev. Biochem., 54:897-930 (1985). PTK enzymes exhibit highspecificity for tyrosine, and ordinarily do not phosphorylate serine,threonine, or hydroxyproline.

More than 75 members of the PTPase family have been identified ineukaryotes, prokaryotes, and even viruses. Tonks and Neel, Cell87:365-368. Protein tyrosine phosphatases (PTPases) were originallyidentified and purified from cell and tissue lysates using a variety ofartificial substrates, and therefore their natural functions andsubstrates were not obvious. However, their roles in cellular processesincluding cell-cell contact and cell adhesion, and growth factor andantigen signaling events, have begun to be elucidated.

PTPases are generally grouped into two categories: those which have bothan extracellular domain and an intracellular catalytic domain, thereceptor PTPases (R-PTPases); and those which are entirelyintracellular. For R-PTPases much effort has been directed atdetermining the function of the extracellular domain. Most of theR-PTPases contain extracellular domains which are structurally similarto domains found in known adhesion molecules; these domains includefibronectin type III repeats, immunoglobulin domains, and cadherinextracellular repeats. See generally Brady-Kalnay and Tonks, Curr. Opin.Cell. Biol. 7:650-657 (1995); Streuli, Curr. Opin. Cell. Biol. 8:182-188(1996). This homology with proteins known to be involved in adhesionsuggested a role for these R-PTPase in regulating or mediating adhesionevents. For several of the R-PTPases, this has now been demonstrated.

Cells form specialized structures at the sites of cell-cell contact(adherens junctions) and cell-extracellular matrix contact (focaladhesion). Multiple signal transduction molecules are recruited to thesesites, including several PTK's; and these sites are characterized byincreased protein tyrosine phosphorylation. These sites are impermanent,and are created and destroyed as required for cell mobility. As enhancedtyrosine phosphorylation is characteristic of the formation of adherensjunctions and focal adhesions, it is likely that protein tyrosinedephosphorylation by PTPases serves to regulate the creation anddestruction of the sites. Supporting this, several studies have shownthat treatment with a general PTPase inhibitor (vanadate) resulted inincreased focal adhesion formation and increased cell spreading. Volberget al., The EMBO J. 11:1733-1742 (1992); Bennett al,. J. Cell Sci.106:891-901 (1993). Importantly, the broadly-expressed LAR R-PTPase hasbeen demonstrated to localize to focal adhesions, apparently via theLAR-interacting protein LIP.1. Serra-Pages et al., The EMBO J.14:2827-2838 (1995). As PTPδ and PTPσ, both R-PTPases, also associatewith LIP.1 [Pulido et al., Proc. Natl. Acad. Sci. 92:11686-11690(1995)], it is likely that these two phosphatases can also localize tofocal adhesions. Most significantly, LAR only localized to the portionof the focal adhesion which is proximal to the nucleus, and is thoughtto be undergoing disassembly. Thus it is likely that these phosphatasesact to negatively regulate focal adhesion formation, acting to enhancethe destruction of the focal adhesion site.

R-PTPases may also act to positively regulate adhesion. Adherensjunctions contain, among others, adhesion receptors termed cadherinswhich mediate cell-cell contact through homophilic binding; thecadherins associate with α-, β-, and γ-catenins, intracellular proteinswhich interact with cortical actin. Association between cadherins andcatenins serves to stabilize the adherens junction and to strengthencell-cell contact. See generally Cowin, Proc. Natl. Acad. Sci.91:10759-10761 (1994). Association of cadherin with β-catenin isdecreased by tyrosine phosphorylation of β-catenin [Kinch el al., J.Cell. Biol. 130:461-471 (1995); Behrens et al., J. Cell. Biol.120:757-766 (1993)]; moreover, treatment with the PTPase inhibitorvanadate inhibits cadherin-dependent adhesion [Matsuyoshi et al., J.Cell. Biol. 118:703-714 (1992)]. Collectively, these data indicate thatPTPase activity is critical in maintaining cadherin-mediated cellaggregation. The R-PTPases PTPμ and PTPκ associate intracellularly withcadherins, and colocalize with cadherins and catenins to adherensjunctions [Brady-Kalnay et al., J. Cell. Biol. 130:977-986 (1995); Fuchset al., J. Biol. Chem. 271:16712-16719 (1996)], thus PTPμ and PITκ arelikely to enhance cadherin function by limiting catenin phosphorylation.

In addition to their catalytic function in regulating adhesion events,several R-PTPases have direct roles in mediating adhesion through theirextracellular domains. PTPκ and PTPμ mediate cellular aggregationthrough homophilic binding [Brady-Kalnay et al., J. Cell. Biol.122:961-972 (1993); Gebbink et al., J. Biol. Chem. 268:16101-16104(1993); Sap et al,. Mol. Cell. Biol. 14:1-9 (1994)]. The neuronal PTPζ(which has also been called R-PTPβ) binds to contactin, a neuronal cellrecognition molecule; binding of PTPζ to contactin increases celladhesion and neuritc outgrowth. Peles et al., Cell 82:251-260 (1995). Asecreted splice variant of PTPζ (also known as phosphacan) binds theextracellular matrix protein tenascin [Barnea et al. J. Biol. Chem.269:14349-14352 (1994)], and the neural cell adhesion molecules N-CAMand Ng-CAM [Maurel et al., Proc. Natl. Acad. Sci. 91:2512-2516 (1994)].As the expression of PTPζ is restricted to radial glial cells in thedeveloping central nervous system, which are though to form barriers toneuronal migration during embryogenesis, it is likely that theinteraction of PTPζ with contactin, tenascin, N-CAM, and/or Ng-CAM actsto regulate neuronal migration. This has been demonstrated for a relatedR-PTPase, DLAR, in Drosophila [Krueger et al. Cell 84:611-622 (1996)].

Because tyrosine phosphorylation by PTK enzymes usually is associatedwith cell proliferation, cell transformation and cell differentiation,it was assumed that PTPases were also associated with these events. Forseveral of the intracellular PTPases, this function has now beenverified.

SHP1 (which has also been called SHPTP1, SHP, HCP, and PTP-1C [seeAdachi et al., Cell 85:15 (1996)]), an intracellular PTPase whichcontains two amino-terminal phosphotyrosyl binding Src Homology 2 (SH2)domains followed by the catalytic PTPase domain, has been demonstratedto be an important negative regulator of growth factor signaling events.See generally Tonks and Neel, supra: Streuli, supra. In mice, loss ofSHP1 function (the motheaten and viable motheaten phenotypes) causesmultiple hematopoietic defects resulting in immunodeficiency and severeautoinmmunity; culminating in lethality by 2-3 weeks or 2-3 monthsdepending on the severity of SHP1 deficiency. Although these mice havereduced numbers of hematopoietic cells, suggesting defects indevelopment and maturation, those cells which survive and enter theperiphery are characterized by hyper-responsiveness to growth factorsand antigen. This observation suggested a role for SHP1 in negativeregulation of hematopoietic signaling events.

This has now been well established for the erythropoietin receptor(EpoR), a member of the cytokine receptor family (which also includesthe receptors for interleukins 2, 3, 4, 5, 6, 7; granulocyte-macrophagecolony stimulating factor, and macrophage colony stimulating factor).SHP1 associates via its SH2 domains with tyrosine-phosphorylated EpoR,causing dephosphorylation and inactivation of the EpoR-associated Januskinase 2 and termination of the cellular response to erythropoietin.Klingmuller et al., Cell 80:729-738 (1995). Mutation of the tyrosine onthe EpoR to which SHP1 binds results in enhanced cell proliferation toerythropoietin in vitro [Klingmuller, supra]. In humans, mutation of theEpoR resulting in loss of association with SHP1 causes autosomaldominant benign erythrocytosis, which is characterized by increasednumbers of erythrocytes in the periphery and increased hematocrit. de laChapelle et al., Proc. Natl. Acad. Sci. 90:4495-4499 (1993).

SHP1 also appears to be a negative regulator of the cellular response tocolony stimulating factor-1 (CSF-1, a major macrophage mitogeniccytokine), as cells from viable motheaten and motheaten mice, which havereduced or absent SHP1 function, are hyper-resporsive to CSF-1 in vitro.Reduced SHP1 expression also results in increased cellular response tointerleukin 3 [Yi et al., Mol. Cell. Biol. 13:7577-7586 (1993)].Collectively, these observations suggest that SHP1 functions to limitthe a cellular response to cytokines and growth factors by reversing thetyrosine phosphorylation of key signaling intermediates in thesepathways.

PTPases appear to play a homologous role in the insulin signalingpathway. Treatment of adipocytes with the PTPase inhibitor vanadateresults in increased tyrosine phosphorylation and tyrosine kinaseactivity of the insulin receptor (InsR), and enhances or mimics thecellular effects of insulin including increased glucose transport. See,e.g., Shisheva and Shechter, Endocrinology 133:1562-1568 (1993); Fantus,et al., Biochemistry 28:8864-8871 (1989); Kadota, et al., Biochem.Biophys. Res. Comm. 147:259-266 (1987); Kadota, et al., J. Biol. Chem.262:8252-8256 (1987). Transiently induced reduction in expression of twoPTPases, the intracellular PTPase PTP-1B and the R-PTPase LAR, resultedin similar increases in the cellular response to insulin. Kulas, et al.,J. Biol. Chem. 270:2435-2438 (1995); Ahmad et al., J. Biol. Chem.270:20503-20508 (1995). Conversely, increased cellular expression ofseveral PTPases (PTPα, PTPε, CD45) in vitro has been demonstrated toresult in diminished InsR signaling [see. e.g., Moller, et al., J. Biol.Chem. 271:23126-23131 (1995); Kulas et al., J. Biol. Chem. 271:755-760(1996)]. Finally, increased expression of LAR was observed in adiposetissue from obese human subjects [Ahmad, et al., J. Clin. Invest.95:2806-2812 (1995)]. These data provide clear evidence that PTPasesnegatively regulate the insulin signaling pathway.

While many of the PTPases function to negatively regulate cellularmetabolism and response, it is becoming increasingly evident thatPTPases provide important positive signaling mechanisms as well. Perhapsthe beat example of such a positive regulator is the hematopoieticR-PTPasc CD45. See generally Streuli, supra; Okumura and Thomas, supra;Trowbridge, Annu. Rev. Immunol. 12:85-116 (1994). CD45 is abundantlyexpressed on the cell surface of all nucleated hematopoietic cells. inseveral alternative splice variants. T and B lymphocytes which lack CD45expression are incapable of responding normally to antigen, suggestingthat CD45 is required for antigen receptor signaling. Geneticallyengineered mice which lack expression of CD45 exhibit severe defects inT lymphocyte development and maturation, indicating an additional rolefor CD45 in thymopoiesis. The major substrates for CD45 appear to bemembers of the Src family of PTK's, particularly Lck and Fyn, whosekinase activity is both positively and negatively regulated by tyrosinephosphorylation. Lck and Fyn isolated from CD45-deficient cells arehyperphosphorylated on negative regulatory tyrosine residues, and theirPTK activity is reduced. As CD45 can dephosphorylate and activatepurified Lck and Fyn in vitro, these data suggest that CD45 maintainsthe activity of Lck and Fyn in vivo through dephosphorylation of thesenegative regulatory tyrosines and that this is an important mechanismfor maintaining lymphocyte homeostasis.

A second PTPase which is now believed to play an important positive rolein signal transduction is the intracellular. SH2-domain-containing SHP2(which las also been called SHPTP-2, SHPTP-3, syp, PTP2c, and PTP-1D[Adachi, et al., supra]). See generally Saltiel, Am. J. Physiol.270:E375-385 (1996); Draznin, Endocrinology 137:2647-2648. SHP2associates, via its SH2 domains, with the receptor for platelet-derivedgrowth factor (PDGF-R), the receptor for epidermal growth factor(EGF-R). with the insulin receptor, and with the predominant substrateof the lnsR. insulin receptor substrate 1 (IRS1). Bennett, et al., Proc.Natl. Acad .Sci. 91:7335-7339 (1994); Case, et al., J. Biol. Chem.269:10467-10474 (1994); Kharitonenkov, et al., J. Biol. Chem.270:29189-29193 (1995); Kuhne, et al., J. Biol. Chem. 268:11479-11481(1993). SHP2 PTPase activity is required for cellular response to EGFand insulin as competitive expression of inactive forms of SHP2 resultsin diminished signaling events and reduced cellular responses to EGF andinsulin. Milarski and Saltiel, J. Biol. Chem. 269:21239-21243 (1994);Xiao et al., J. Biol. Chem. 269:21244-21248 (1994); Yamauchi el al.,Proc. Natl. Acad. Sci. 92:664-668 (1995). The relevant substrate(s) forthe PTPase domain of SHP2 is not known.

Due to the fundamental role that PTPases play in normal and neoplasticcellular growth and proliferation. A need exists in the art for agentscapable of modulating PTPase activity. On a fundamental level, suchagents are useful for elucidating the precise role of protein tyrosinephosphatases and kinases in cellular signalling pathways and cellulargrowth and proliferation. See generally MacKintosh and MacKintosh, TIBS,19:444-448 (1994).

More importantly, modulation of PTPase activity has important clinicalsignificance. For example, PTP-1B overexpression has been correlatedwith breast and ovarian cancers [Weiner et al., J. Natl. Cancer Inst.,86:372-8 (1994); Weiner et al., Am J. Obstet. Gynecol., 170:1177-883(1994)], and thus agents which modulate PTP-1B activity would be helpfulin elucidating the role of PTP-1B in these conditions and for thedevelopment of effective therapeutics against these disease states. Theimportant role of CD45 in hematopoietic development and T lymphocytefunction likewise indicates a therapeutic utility for PTPase inhibitorsin conditions that are associated with autoimmune disease, and as aprophylaxis for transplant rejection. The antibiotic suramin, which alsoappears to possess anti-neoplastic indications, has recently been shownto be a potent, irreversible, non-competitive inhibitor of CD45. SeeGhosh and Miller, Biochem. Biophys. Res. Comm. 194:36-44 (1993). Thenegative regulatory effects of several PTPases on signaling throughreceptors for growth factors and cytokines, which are implicated innormal cell processing as well as discase states such as cancer andatherosclerosis, also indicate a therapeutic potential for PTPaseinhibitors in diseases of hematopoietic origin.

The PTPase Yop2b is an essential virulence determinant in the pathogenicbacterium Yersinia, responsible for bubonic plague. Bliska et al., Proc.Natl. Acad. Sci. USA. 88:1187-91 (1991), and thus an antimicrobialindication exists for PTPase inhibitor compounds, as well.

PTPases have been implicated in diabetic conditions. The relevents withone family of PTPase inhibitors, vanadium derivatives, indicate atherapeutic utility for such compounds as oral adjuvants or asalternatives to insulin for the treatment of hyperglycemia, See Posneret al., J. Biol. Chem., 269:4596-4604 (1994). However, suchmetal-containing PTPase inhibitors act in a fairly nonspecific fashionand act with similar potencies against all PTPase enzymes.

In addition to vanadium derivatives, certain organic phosphotyrosineminmetics are reportedly capable of competitively inhibiting PTPasemolecules when such mimetics are incorporated into polypeptideartificial PTPase substrates of 6.11 amino acid residues. For example, a“natural” (phosphorylated tytosine) PTPase substrate, which may bedepicted by the Formula:

has been mimicked by eleven-mer oligopeptides containing phosphonomethylphenvlalanine (Pmp), as depicted by the schematic Formula:

See Chatterjee et al., “Phosphopeptide substrates and phosphonopeptideinhibitors of protein tyrosine phosphatases,” in Pepindes: Chemistry andBiology (Rivier and Smith, Eds.), 1992, Escom Science Publishers;Leiden, Netherlands, pp. 553-55; Burke et al., Biochemistry, 33:6490-94(1994). More recently, Burke et al., Biochem. Biophys. Res. Comm.204(1):129-134 (1994) reported that a particular hexameric peptidesequence comprising a Pmp moiety or, more preferably, aphosphonodifluoromethyl phenylalanine (F₂Pmp) moiety, as depicted by theschematic Formula:

competitively inhibited PTP-1B. However, such hexapeptide inhibitorsnonetheless possess drawbacks for PTPase modulation in vivo. Moreparticularly, the hexapeptide inhibitors described by Burke et al. aresufficiently large and anionic to potentially inhibit efficientmigration across cell membranes, for interaction with the catalyticdomains of transmembrane and intracellular PTPase enzymes which liewithin a cell membrane. A need exists for small, organic-molecule basedPTPase inhibitors having fewer anionic moieties to facilitate migrationacross cell membranes.

For all of the foregoing reasons, a need exists in the art for novelcompounds effective for modulating, and especially inhibiting, thephosphatase activity of protein tyrosine phosphatase molecules.

SUMMARY OF THE INVENTION

The invention provides compounds and derivatives thereof useful formodulating, wad especially inhibiting, the phospbatase activity of oneor more protein tyrosine phosphatase (PTPase) and/or dual specificityphosphatase enzymes. In one aspect, the present invention relates tocompounds having the general structure shown in Formula (A1):

wherein R′, R″, R′″, X and Y are defined below. The inventions furtherprovides salts, esters, prodrugs, solvates, and the like of thecompounds, and compositions comprising these compounds.

DEFINITIONS

In the specification and claims the term “derivatives” means: arylacrylic acids with structure depicted in Formula (A1) havingsubstitution (with, e.g., hydrogen, hydroxy, halo, amino, carboxy,nitro, cyano, methoxy, etc.) at one or more atoms of the aryl ring.Moreover, “derivatives” includes compounds of the Formula (A1) havingsubstitution at the alkene carbons with, e.g., an electron withdrawinggroup (e.g., Cl, F, Br, CF₃, phenyl) or an electron donating group(e.g., CH₃, alkoxy).

As used herein, the term “attached” signifies a stable covalent bond,certain preferred points of attachment being apparent to those skilledin the art.

The terms “halogen” or “halo” include fluorine, chlorine, bromine, andiodine.

The term “alkyl” includes C₁-C₁₁ straight chain saturated and C₂-C₁₁unsaturated aliphatic hydrocarbon groups, C₁-C₁₁ branched saturated andC₂-C₁₁ unsaturated aliphatic hydrocarbon groups, C₃-C₈ cyclic saturatedand C₅-C₈ unsaturated aliphatic hydrocarbon groups, and C₁-C₁₁ straightchain or branched saturated and C₂-C₁₁ straight chain or branchedunsaturated aliphatic hydrocarbon groups substituted with C₃-C₈ cyclicsaturated and unsaturated aliphatic hydrocarbon groups having thespecified number of carbon atoms. For example, this definition shallinclude but is not limited to methyl (Me), ethyl (Et), propyl (Pr),butyl (Bu), pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,ethenyl, propenyl, butenyl, penentyl, hexenyl, heptenyl, octenyl,nonenyl, decenyl, undecenyl, isopropyl (i-Pr), isobutyl (i-Bu),tert-butyl (t-Bu), sec-butyl (s-Bu), isopentyl, neopentyl, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl,methylcyclopropyl, ethylcyclohexenyl, butenylcyclopentyl, and the like.

The term “substituted alkyl” represents an alkyl group as defined abovewherein the substitutents are independently selected from halo, cyano,nitro, trihalomethyl, carbamoyl, C₀₋₁₁alkyloxy, arylC₀₋₁₁alkyloxy,C₀₋₁₁alkylthio, arylC₀₋₁₁alkylthio, C₀₋₁₁alkylamino,arylC₀₋₁₁alkylamino, di(aryC₀₋₁₁alkyl)amino, C₁₋₁₁alkylcarbonyl,arylC₁₋₁₁alkylcarbonyl, C₁₋₁₁alkylcarboxy, arylC₁₋₁₁alkylcarboxy,C₁₋₁₁alkylcarbonylamino, aryl C₁₋₁₁alkylcarbonylamino, tetrahydrotiryl,morpholinyl, piperazinyl, hydroxypyronyl, —C₀₋₁₁alkylCOOR₁,—C₀₋₁₁alkylCONR₂R₃ wherein R₁, R₂ and R₃ are independently selected fromhydrogen, C₁-C₁₁alkyl, arylC₀-C₁₁alkyl, or R₂ and R₃ are taken togetherwith the nitrogen to which they are attached forming a cyclic systemcontaining 3 to 8 carbon atoms with at least one C₁-C₁₁alkyl,arylC₀-C₁₁alkyl substituent.

The term “alkyloxy” (e.g. methoxy, ethoxy, propyloxy, allyloxy,cyclohexyloxy) represents an alkyl group as defined above having theindicated number of carbon atoms attached through an oxygen bridge. Theterm “alkyloxyalkyl” represents an alkyloxy group attached through analkyl group as defined above having the indicated number of carbonatoms.

The term “alkylthio” (e.g. methylthio, ethylthio, propylthio,cyclohexenylthio and the like) represents an alkyl group as definedabove having the indicated number of carbon atoms attached through asulfur bridge. The term “alkylthioalkyl” represents an alkylthio groupattached through an alkyl group as defined above having the indicatednumber of carbon atoms.

The term “alkylamino” (e.g. methylamino, diethylamino, butylamino,N-propyl-N-hexylamino, (2-cyclopentyl)propylamino, hxenylamino,pyrrolidinyl, piperidinyl and the like) represents one or two alkylgroups as defined above having the indicated number of carbon atomsattached through an amine bridge. The two alkyl groups maybe takentogether with the nitrogen to which they are attached forming a cyclicsystem containing 3 to 11 carbon atoms with at least one C₁-C₁₁alkyl,arylC₀-C₁₁alkyl substituent. The term “alkylaminoalkyl” represents analkylamino group attached through an alkyl group as defined above havingthe indicated number of carbon atoms.

The term “alkylcarbonyl” (e.g. cyclooctylcarbonyl, pentylcarbonyl,3-hexenylcarbonyl) represents an alkyl group as defined above having theindicated number of carbon atoms attached through a carbonyl group. Theterm “alkylcarbonylalkyl” represents an alkylcarbonyl group attachedthrough an alkyl group as defined above having the indicated number ofcarbon atoms.

The term “alkylcarboxy” (e.g. heptylcarboxy, cyclopropylcarboxy,3-pentenylcarboxy) represents an alkylcarbonyl group as defined abovewherein the carbonyl is in turn attached through an oxygen. The term“alkylcarboxyalkyl” represents an alkylcarboxy group attached through analkyl group as defined above having the indicated number of carbonatoms.

The term “alkylcarbonylamino” (e.g. hexylcarbonylamino,cyclopentylcarbonyl-aminomethyl, methylcarbonylaminophenyl) representsan alkylcabonyl group as defined above wherein the carbonyl is in turnattached through the nitrogen atom of an amino group. The nitrogen groupmay itself be substituted with an alkyl or aryl group. The term“alkylcarbonylaminoalkyl” represents an alkylcarbonylamino groupattached through an alkyl group as defined above having the indicatednumber of carbon atoms. The nitrogen group may itself be substitutedwith an alkyl or aryl group.

The term “aryl” represents an unsubstituted, mono-, di- ortrisubstituted monocyclic, polycyclic, biaryl and heterocyclic aromaticgroups covalently attached at any ring position capable of forming astable covalent bond, certain preferred points of attachment beingapparent to those skilled in the art (e.g., 3-indolyl, 4-imidazolyl).The aryl substituents are independently selected from the groupconsisting of halo, nitro, cyano, trihalomethyl, hydroxypyronyl,C₁₋₁₁alkyl, arylC₁₋₁₁alkyl, C₀₋₁₁alkyloxyC₀₋₁₁alkyl,arylC₀₋₁₁alkyloxyC₀₋₁₁alkyl, C₀₋₁₁alkylthioC₀₋₁₁alkyl,arylC₀₋₁₁alkylthioC₀₋₁₁alkyl, C₀₋₁₁alkylaminoC₀₋₁₁alkyl,arylC₀₋₁₁alkylaminoC₀₋₁₁alkyl, di(arylC₁₋₁₁alkyl)aminoC₀₋₁₁alkyl,C₁₋₁₁alkylcarbonylC₀₋₁₁alkyl, arylC₁₋₁₁alkylcarbonylC₀₋₁₁alkyl,C₁₋₁₁alkylcarboxyC₀₋₁₁alkyl, arylC₁₋₁₁lalkylcarboxyC₀₋₁₁alkyl,C₁₋₁₁alkylcarbonylaminoC₀₋₁₁alkyl,arylC₁₋₁₁alkylcarbonylaminoC₀₋₁₁alkyl, —C₀₋₁₁alkylCOOR₄,—C₀₋₁₁alkylCONR₅R₆ wherein R₄, R₅ and R₆ are independently selected fromhydrogen, C₁-C₁₁alkyl, arylC₀-C₁₁alkyl, or R₅ and R₆ are taken togetherwith the nitrogen to which they are attached forming a cyclic systemcontaining 3 to 8 carbon atoms with at least one C₁-C₁₁alkyl,arylC₀-C₁₁alkyl substituent.

The definition of aryl includes but is not limited to phenyl, biphenyl,naphthyl, dihydronaphthyl, tetrahydronaphthyl, indenyl, indanyl,azulenyl, anthryl, phenanthryl, fluorenyl, pyrenyl, thienyl,benzothienyl, isobenzothienyl, 2,3-dihydrobenzothienyl, furyl, pyranyl,benzofuranyl, isobenzofuranyl, 2,3-dihydrdbenzofuranyl, pyrrolyl,indolyl, isoindolyl, indolizinyl, indazolyl, imidazolyl, benzimidazolyl,pyridyl, pyrazinyl, pyradazinyl, pyrimidinyl, triazinyl, quinolyl,isoquinolyl, 4H-quinolizinyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, chromanyl, benzodioxolyl,piperonyl, purinyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl,isothiazolyl, benzthiazolyl, oxazolyl, isoxazolyl, benzoxazolyl,oxadiazolyl, thiadiazolyl.

The term “arylalkyl” (e.g. (4-hydroxyphenyl)ethyl,(2-aminonaphthyl)hexenyl, pyridylcyclopentyl) represents an aryl groupas defined above attached through an alkyl goup as defined above havingthe indicated number of carbon atoms.

The term “arylcarbonyl” (e.g. 2-thiophenylcarbonyl,3-methoxyanthrylcarbonyl, oxazolylcarbonyl) represents an aryl group asdefined above attached through a carbonyl group.

The term “arylalkylcarbonyl” (e.g. (2,3-dimethoxyphenyl)propylcarbonyl,(2-chloronaphthyl)pentenylcarbonyl, imidazolylcyclopentylcarbonyl)represents an arylalkyl group as defined above wherein the alkyl groupis in turn attached through a carboryl.

The term “signal transduction” is a collective term used to define allcellular processes that follow the activation of a given cell or tissue.Examples of signal transduction include but are not in any way limitedto cellular events that are induced by polypeptide hormones and growthfactors (e.g. insulin, insulin-like growth factors I and II, growthhormone, epidermal growth factor, platelet-derived growth factor),cytokines (e.g. interleukines), extracellular matrix components, andcell-cell interactions.

Phosphotyrosine recognition units/tyrosine phosphate recognitionunits/phosphotyrosine recognition units are defined as areas or domainsof proteins or glycoproteins that have affinity for molecules containingphosphorylatad tyrosine residues (pTyr). Examples of pTyr recognitionunits include but are not in any way limited to: PTPases, SH2 domainsand PTB domains.

PTPases are defined as enzymes with the capacity to dephosphorylatepTyr-containing proteins or glycoproteins. Examples of PTPases includebut are not in any way limited to: intracellular PTPases (e.g. PTP-1B,TC-PTP, PTP-1C, PTP-1D, PTP-D1, PTP-D2), receptor-type PTPases (e.g.PTPα, PTPε, PTPβ, PTPγ, CD45, PTPκ, PTPμ), dual specificity phosphatases(e.g. VH1, VHR, cdc25) and other PTPases such as LAR, SHP-1, SHP-2,PTP-1H, PTPMEGI, PTP-PEST, PTPζ, PTPS31, IA-2 and HePTP and the like.

Modulation of cellular processes is defined as the capacity of compoundsof the invention to 1) either increase or decrease ongoing, normal orabnormal, signal transduction, 2) initiate normal signal transduction,and 3) initiate abnormal signal a transduction.

Modulation of pTyr-mediated signal transduction/modulation of theactivity of molecules with pTyr recognition units is defined as thecapacity of compounds of the invention to 1) increase or decrease theactivity of proteins or glycoproteins with pTyr recognition units (e.g.PTPases, SH2 domains or PTB domains) or to 2) decrease or increase theassociation of a pTyr-containing molecule with a protein or glycoproteinwith pTyr recognition units either via a direct action on the pTyrrecognition site or via an indirect mechanism. Examples of modulation ofpTyr-mediated signal transduction/modulation of the activity ofmolecules with pTyr recognition units, which are not intended in any waylimiting to the scope of the invention claimed, are: a) inhibition ofPTPase activity leading to either increased or decreased signaltransduction of ongoing cellular processes; b) inhibition of PTPaseactivity leading to initiation of normal or abnormal cellular activity;c) stimulation of PTPase activity leading to either increased ordecreased signal transduction of ongoing cellular processes; d)stimulation of PTPase activity leading to initiation of normal orabnormal cellular activity; e) inhibition of binding of SH2 domains orPTB domains to proteins or glycoproteins with pTyr leading to increaseor decrease of ongoing cellular processes; f) inhibition of binding ofSH2 domains or PTB domains to proteins or glycoproteins with pTyrleading to initiation of normal or abnormal cellular activity.

A subject is defined as any mammalian species, including humans.

DETAILED DESCRIPTIONS

This application relates to compounds having the general structure shownin Formula (A1):

wherein

(i) R′ and R″ are independently selected from the group consisting ofhydrogen, halo, cyano, nitro, trihalomethyl, alkyl, arylalkyl,

(ii) R′″ is selected from the group consisting of hydrogen, alkyl,substituted alkyl, aryl, arylalkyl

(iii) X is aryl,

(iv) Y is selected from hydrogen or

wherein (*)indicates a potential point of attachment to X and all otherpositions are substituted as described below.

(1) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A2):

wherein at least one of R₁, R₂ and R₃ substituents has the generalstructure depicted in Formula (B)

wherein R′, R″, R′″ and X are defined as above in Formula (A1), andwherein the remaining of R₁, R₂ and R₃ are independently selected fromthe goup consisting of hydrogen, alkyl, substituted alkyl, aryl,arylalkyl.

(2) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A3):

wherein at least one of R₁, R₂ and R₃ substituents has the generalstructure depicted in Formula (B)

wherein R′, R″, R′″ and X are defined as above in Formula (A1), adwherein the remaining of R₁, R₂ and R₃ are independently selected fromthe group consisting of: hydrogen, alkyl, substituted alkyl,alkylcarbonyl, substituted alkylcarbonyl, aryl, arylalkyl, arylcarbonyl,arylalkylcarbonyl.

(3) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A4):

wherein at least one of R₁, R₂ substituents has the general structuredepicted in Formula (B)

wherein R′, R″, R′″ and X are defined as above in Formula (A1), andwherein the remaining of R₁, R₂ is defined as above in Formula (A2).

(4) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A5):

wherein at least one of R₁ and R₂ substituents has the general structuredepicted in Formula (B)

wherein R′, R″, R′″ and X are defined as above in Formula (A1), andwherein the remaining of R₁ and R₂ is defined as above in Formula (A2).

(5) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A6):

wherein at least one of R₁, R₂, R₃ and R₄ substituents has the generalstructure depicted in Formula(B)

wherein R′, R″, R′″ and X are defined as above in Formula (A1), andwherein the remaining of R₁, R₂, R₃ and R₄ have the same definition asR₁, R₂ and R₃ in Formula (A2), with the proviso that when R₃ and R₄ areselected from substituted phenyl or substituted furyl then the phenyland furyl substituents exclude hydroxy, halo, trifluoromethyl C₁₋₆alkyl,C₁₋₆alkyloxy, C₁₋₆alkylthio, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino,phenylC₁₋₆alkylamino and di(phenylC₁₋₆alkyl)amino.

(6) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A6):

wherein R₄ is selected from —COR₅, —COOR₆, —CONR₇R₈ wherein R₅ thru R₈are selected from hydrogen, alkyl, substituted alkyl, aryl, arylalkyl,or R₇ and R₈ are taken together with the nitrogen to which they areattached forming a cyclic system containing 3 to 8 carbon atoms with atleast one alkyl, aryl, arylalkyl substituent, and wherein at least oneof R₁, R₂, and R₃ substituents has the general structure depicted inFormula (B)

wherein R′, R″, R′″ and X are defined as above in Formula (A1), andwherein the remaining of R₁, R₂ and R₃ are defined as above in Formula(A2).

(7) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A6):

wherein R₁, R₂, R₃ and R₄ we defined as above in (6).

(8) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A7):

wherein R₂ is selected from —COR₅, —COOR₆, —CONR₇R₈ wherein R₅ thru R₈are defined as above in (6) and wherein at least one of R₁ and R₃substituents has the general structure depicted in Formula (B)

wherein R′, R″, R′″ and X are defined as above in Formula (A1), andwherein the remaining of R₁ and R₃ are defined as above in Formula (A2).

(9) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A8):

wherein at least one of R₁ and R₂ substituents has the general structuredepicted in Formula (B)

wherein R′, R″, R′″ and X are defined as above in Formula (A1), andwherein the remaining of R₁ and R₂ is defined as above in Formula (A2),and wherein m is an integer between 0 and 4 and each R₃ is independentlyselected from the group consisting of halo, nitro, cyano, trihalomethyl,hydroxypyronyl, alkyl, arylalkyl, C₀₋₁₁alkyloxyC₀₋₁₁alkyl,arylC₀₋₁₁alkyloxyC₀₋₁₁alkyl, C₀₋₁₁alkylthioC₀₋₁₁alkyl,arylC₀₋₁₁alkylthioC₀₋₁₁alkyl, C₀₋₁₁alkylaminoC₀₋₁₁alkyl,arylC₀₋₁₁alkylaminoC₀₋₁₁alkyl, di(arylC₁₋₁₁alkyl)aminoC₀₋₁₁alkyl,C₁₋₁₁alkylcarbonylC₀₋₁₁alkyl, arylC₁₋₁₁alkylcarbonylC₀₋₁₁alkyl,C₀₋₁₁alkylcarboxyC₀₋₁₁alkyl, arylC₀₋₁₁alkylcarboxyC₀₋₁₁alkyl,C₁₋₁₁alkylcarbonylaminoC₀₋₁₁alkyl,arylC₁₋₁₁alkylcarbonylaminoC₀₋₁₁alkyl,—C₀₋₁₁alkylCOOR₄,—C₀₋₁₁alkylCONR₅R₆ wherein R₄, R₅ and R₆ areindependently selected from hydrogen, C₁-C₁₁alkyl, arylC₀-C₁₁alkyl, orR₅ and R₆ are taken together with the nitrogen to which they areattached forming a cyclic system containing 3 to 8 carbon atoms with atlet one C₁-C₁₁ alkyl, arylC₀-C₁₁ alkyl substituent.

(10) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A8).

wherein R₁ is selected from —COR₅, —COOR₆, —CONR₇R₈ wherein R₅ thru R₈are defined as above in (6) and wherein R₂ has the general structuredepicted in Formula (B)

wherein R′, R″, R′″ and X are defined as above in Formula (A1), andwherein m is an integer between 0 and 4 and each R₃ is defined as abovein (9).

(11) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A9):

wherein m is an integer between 0 and 3 and wherein R₁, R₂ each R₃ isdefined as above in (9).

(12) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A9):

wherein either R₁ or R₂ is selected from —COR₅, —COOR₆, —CONR₇R₈ whereinR₅ thru R₈ are defined as in (6) and wherein the remainder of R₁ and R₂is defined as above in (9), and wherein m is an integer between 0 and 3and each R₃ is defined as above in (9).

(13) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structure Formuladepicted in (A10):

wherein Z₁ and Z₂ are independently selected from the group consistingof OR₃, SR₄, NR₅R₆ and wherein at least one of R₁, R₂ substituents hasthe general structure depicted in Formula (B)

wherein R′, R″, R′″ and X are defined as above in Formula (A1), andwherein the remaining of R₁, R₂ is defined as above in Formula (A2), andwherein R₃, R₄, R₅, R₆ are independently selected from hydrogen, alkyl,substituted alkyl, alkylcarbonyl, substituted alkylcarbonyl, aryl,arylalkyl, arylcarbonyl, arylalkycarbonyl.

(14) According to the invention, a class of preferred PTPaseactivity-modulating compounds have the general structural Formuladepicted in (A11):

wherein at least one of R₁, R₂, and R₃ substituents has the generalstructure depicted in Formula (B)

wherein R′, R″, R′″ and X are defined as above in Formula (A1), andwherein the remaining of R₁, R₂ and R₃ are defined as above in Formula(A2).

Preferred compositions of the invention include compositions comprisingcompounds as defined above in structural formula (A1), (A2), (A3), (A4),(A5), (A6), (A7), (A8), (A9), (A10), (A11) (or pharmaceuticallyacceptable salts, prodrugs, esters, or solvates of these compounds) inadmixture with a pharmaceutically acceptable diluent, adjuvant, orcarrier.

Provided according to the invention, therefore, are novel compoundswhich modulate the activity of PTPase or other molecules with pTyrrecognition units) as well as previously known aryl acrylic acidcompounds which modulate the activity of PTPase or other molecules withpTyr recognition unit(s).

Another aspect of the present invention provides compositions comprisingPTPase modulating compounds of the invention suitable for administrationto a mammalian host.

In a preferred embodiment the compounds of the invention act asinhibitors of PTPases, e.g. protein tyrosine phosphatases involved inthe regulation of tyrosine kinase signaling pathways. Preferredembodiments include modulation of receptor-tyrosine kinase signalingpathways via interaction with regulatory PTPases, e.g. the signalingpathways of the insulin receptor, the IGF-I receptor and other membersof the insulin receptor family, the EGF-receptor family, theplatelet-derived growth , factor family, the nerve growth factorreceptor family, the hepatocyte growth factor receptor family, thegrowth hormone receptor family and members of other receptor-typetyrosine kinase families. Further preferred embodiments of the inventionis modulation of non-receptor tyrosine kinase signaling throughmodulation of regulatory PTPases, e.g. modulation of members of the Srckinase family. One type of preferred embodiments of the inventionrelates to modulation of the activity of PTPases that negativelyregulate signal transduction pathways. Another type of preferredembodiments of the inventions relate to modulation of the activity ofPTPases that positively regulate signal transduction pathways.

In a preferred embodiment compounds of the inventions act as modulatorsof the active site of PTPases. In another preferred embodiment thecompounds of the invention modulate the activity of PTPases viainteraction with structures positioned outside the active sites of theenzymes, preferably SH2 domains. Further preferred embodiments includemodulation of signal transduction pathways via binding of the compoundsof the invention to SH2 domains or PTB domains of non-PTPase signalingmolecules.

Other preferred embodiments include use of the compounds of theinvention for modulation of cell-cell interactions as well ascell-matrix interactions.

As a preferred embodiment the compounds of the invention may be used astherapeutics to inhibit PTPases involved in the regulation of theinsulin receptor tyrosine kinase signaling pathway in patients with typeI diabetes, type II diabetes, impaired glucose tolerance, insulinresistance and obesity. Further preferred embodiments include use of thecompounds of the invention for treatment of disorders with general orspecific dysfunction of PTPase activity, e.g. proliferative disordersincluding neoplastic diseases and psoriasis. As another embodiment, thecompounds of the invention may be used in pharmaceutical preparationsfor treatment of osteoporosis.

Preferred embodiments of the invention further include use of compoundsof the invention in pharmaceutical preparations to increase thesecretion or action of growth hormone and its analogs or somatomedinsincluding IGf-I and IGF-2 by modulating the activity of PTPases or othersignal transduction molecules with affinity for phosphotyrosine involvedcontrolling or inducing the action of these hormones or any regulatingmolecule.

To those skilled in the art, it is well known that the current andpotential uses of growth hormone in humans are varied and muti-tudinous.Thus, compounds of the invention can be administered for purposes ofstimulating the release of growth hormone from the pituitary or increaseits action on target tissues thereby leading to similar effects or usesas growth hormone itself. The uses of growth hormone maybe summarized asfollows: stimulation of growth hormone release in the elderly;prevention of catabolic side effects of glucocorticoids; treatment ofosteoporosis, stimulation of the immune system; treatment ofretardation, acceleration of wound healing; accelerating bone fracturerepair; treatment of growth retardation; treating renal failure orinsufficiency resulting in growth retardation; treatment ofphysiological short stature including growth hormone deficient childrenand short stature associated with chronic illness; treatment of obesityand growth retardation associated with obesity; treating growthretardation assumed with the Pader-Willi syndrome and Turner's syndrome;accelerating the recovery and reducing hospitalization of burn patients;treatment of intrauterine growth retardation, skeletal dysplasia,hypercortisolism and Cushings syndrome; induction of pulsatile growthhormone release; replacement of growth hormone in stressed patients;treatment of osteochondro-dysplasis, Noonans syndrome, schizophrenia,depressions, Alzheimer's disease, delayed wound healing and psychosocialdeprivation; treatment of pulmonary dysfunction and ventilatordependency; attenuation of protein catabolic responses after majorsurgery; reducing cachexia and protein loss due to chronic illness suchas cancer or AIDS; treatment of hyperinsulinemia includingnesidio-blastosis; adjuvant treatment for ovulation induction;stimulation of thymic development and prevention of age related declineor thymic function; treatment of immunosuppresed patients; improvementin muscle strength, mobility, maintenance of skin thickness, metabolichomeostasis, renal homeostasis in the frail elderly; stimulation ofosteoblasms, bone remodelling and cartilage growth; stimulation of theimmune system in companion animals and treatment of disorder of aging incompanion animals; growth promotant in livestock and stimulation of woolgrowth in sheep.

The compounds of the invention may be used in pharmaceuticalpreparations for treatment of various disorders of the immune system,either as stimulant or suppresor of normal or perturbed immunefunctions, including autoimmnune reactions. Further embodiments of theinvention for treatment of allergic reactions, e.g. asthma, dermalreactions, conjunctivitis.

In another embodiment, compounds of the invention may be used inpharmaceutical preparations for prevention or induction of plateletaggregation

In yet another embodiment, compounds of the invention may be used inpharmaceutical preparations for treatment of infectious disorders. Inparticular, the compounds of the invention may be used for treatment ofinfectious disorders caused by Yersinia and other bacteria as well asdisorders caused by viruses of other micro-organisms.

Compounds of the invention may additionally be used for treatment orprevention of diseases in animals, including commercially importantanimals.

Also included in the present invention is a process for isolation ofPTPases via affinity purification procedures based on the use ofimmobilized compounds of the invention using procedures well-known totose skilled in the art.

The invention is further directed to a method for detecting the presenceof PTPases in cell or in a subject comprising

(a) contacting said cell or an extract thereof with labeled compounds ofthe invention.

(b) detecting the binding of the compounds of the invention or measuringthe quantity bound, thereby detecting the presence or measuring thequantity of certain PTPases.

The invention further relates to analysis and identification of thespecific functions of certain PTPases by modulating their activity byusing compounds of the invention in cellular assay systems or in wholeanimals.

The invention further provides methods for making compounds (A1), (A2),(A3), (A4), (A5), (A6), (A7), (A8), (A9), (A10), (A11) of the presentinvention having PTPase-modulatory/inhibitory activity. In preferredmethods, compounds of the invention are synthesized in a multi-componentcombinatorial array, which permits rapid synthesis of numerous,structurally related compounds for subsequent evaluation In preferredsynthesis protocols, the acrylic acid moiety of a compound is protectedduring synthesis by, e.g., esterification with a tert-butyl protectinggroup. Thus, a preferred method of making compounds of the inventioncomprises use of a protected acrylic acid reagent and removal of theprotective group by, e.g., treatment of a precursor ester compound withacid. Optionally, such a method includes further esterifying orsalifying the acrylic acid product thereby obtained.

The compounds of formula (A1), (A2), (A3), (A4), (A5), (A6), (A7), (A8),(A9), (A10), (A11) may be prepared by procedures known to those skilledin the art from known compounds or readily preparable intermediates.General synthetic procedures and examples are as follow:

General Method for the Removal of tert-butyl Esters

Unless otherwise stated, tert-butyl esters were converted to theircorresponding carboxylic acids via treatment with a solution of 50%trifluoroacetic acid in dichloromethnne for 1 hour at 23° C. The solventwas removed in vacuo and the residue was azeotroped with toluene oracetonitrile to yield the corresponding carboxylic acid.

General Method for the Synthesis of Compounds (A1) and (A5)

Method 1

By allowing a compound of formula (1) wherein LG is a suitable leavinggroup such as bromo, iodo, or triflate to react with compound of formula(2) wherein Z is hydrogen (Heck reaction: J. Org. Chem., 1977, 42,3903), or trialkyltin (Stille reaction: J. Am. Chem. Soc., 1991, 113,9585), or B(OH)₂ (Suzuki reaction: J. Am. Chem. Soc., 1989, 111, 314)and wherein R′, R″, R′″ and X are defined as above for formula (A1).

These reactions may be carried out neat or in a solvent such asdimethylformamide (DMF), tetahydrofiran (THF), or toluene, in thepresence of a catalyst (e.g. Pd(OAc)₂, Pd(PPh₃)₄, Pd₂dba₃), a ligand(e.g. Ph₃P, Ph₃As, (o-tolyl)₃P) and a base (e.g. K₂CO₃, CsCO₃, Et₃N) attemperatures ranging from 23° C. to 130° C., for 1 to 60 hours.

EXAMPLES

Prepared according to Patel et al (J. Org. Chem., 1977, 42, 3903).

¹H NMR of 3 (400 MHz, CDCl₃) δ 1.5 (s, 9H), 6.4 (d, 1H), 7.6 (m, 3H),8.05 (d, 2H).

Prepared according to Patel et al (J. Org. Chem., 1977, 42, 3903).

¹H NMR of 4 (400 MHz, CDCl₃) δ 1.5 (s, 9H), 6.4 (d, 1H), 7.55 (d, 1H),7.6 (d, 2H), 7.8 (d, 2H), 9.95 (s, 1H).

Prepared according to Patel et al (J. Org. Chem., 1977, 42, 3903).

¹H NMR of 5 (400 MHz, CDCl₃) δ 1.44 (s, 9H), 6.26 (d, 1H), 7.18 (d, 1H),7.56 (d, 1H), 7.74 (d, 1H)

Prepared according to Patel et al (J. Org. Chem. 1977, 42, 3903).

¹H NMR of 6 (400 MHz, CDCl₃) δ (s 18H), 6.42 (d, 2H), 7.6 (m, 6H), 7.9(d, 4H).

Prepared according to Patel et al (J. Org. Chem. 1977, 42, 3903).

¹H NMR of 7 (400 MHz, CDCl₃) δ 1.5 (s, 18H), 6.2 (d, 2H), 7.1 (d, 2H),7.35 (d, 2H), 7.5 (s, 2H), 7.7 (d, 2H).

To 11 g of 4,4′-dibromobenzil (30 mmol, 1.0 equiv), 67 mg of palladium(II) acetate (0.3 mmol, 0.01 equiv), 365 mg of tri-o-tolylphosphine (1.2mmol, 0.04 equiv) was added 200 mL of dimnethylformamide followed by 4.2mL (30 mmol, 1.0 equiv) of triethylamine. The mixture was placed in a100° C. preheated bath and 4.4 mL of tert-butylacrylate (30 mmol, 1.0equiv) in 30 mL of dimethylforamide was added dropwise over 1 hour. Thereaction mixture was heated at 100° C. for 12 hours, cooled to 23° C.and the solvent was removed in vacuo. Ethyl acetate was added and theorganic layer was washed with water and dried over sodium sulfate. Thesolvent was rmoved and the residue (mixture of dibromobenzil, mono andbis-teri-butylacrylate benzil) was recrystallized from hot 30%dichloromethane in hexane. The solid which crashed out (mixture ofdibromobenzil and monotert-butylacrylate benzil) was filtered off andtreated with 20% trifluoroacetic acid in dichloromethane. After 20minutes, the mono-tert-butylacrylat benzil 8 was filtered off and washedwith 20% trifluroacetic acid in dichloromethane (1.4 g isolated). Themother liquor (mixture of mono and bis-tert-butylacrylate benzil) wasrecovered and purified by flash chromatography (ethyl acetate-hexaneeluant) to yield 2.4 g of the mono-tert-butylacrylate dione which wastreated with 20% trifluoroacetic acid in dichloromethane to give 2.2 gof 8. The combined total yield of 8 was 3.6 g (34%). ¹H NMR of 8 (400MHz, d₆-DMSO) δ 6.7 (d, 1H), 7.6 (d, 1H), 7.8 (s, 4H), 7.9 (s, 4H).

¹H NMR of 9 (400 MHz, d₆-DMSO) δ 6.7 (d, 2H), 7.6 (d, 2H), 7.9 (s, 8H).

Prepared according to Patel et al (J. Org. Chem., 197, 42, 3903).

¹H NMR of 10 (400 MHz, CDCl₃-CD₃OD 9:1) δ 1.45 (s, 9H), 6.42 (d, 1H),6.5 (d, 1H), 7.55 (d, 1H), 7.6 (dd, 4H), 7.68 (d, 1H), 7.92 (dd, 4H).

To a solution of 10 (1 equiv) in dichloromethane was added octylamine (1equiv), EDCI (1.3 equiv) and 4-dimethylaminopyridine (0.5 equiv) at 23°C. The solution was stirred overnight, diluted with ethyl acetate,washed with 1N HCl and saturated sodium bicarbonate and dried oversodium sulfate. The residue was purified by flash chromatography (ethylacetate-hexane eluant) and the solvent was removed in vacuo to yieldcompound 11. ¹H NMR of 11 (400 MHz, CDCl₃) δ 0.9 (t, 3H), 1.25 (s br,10H), 1.5 (s, 9H), 1.55 (s br, 2H), 3.35 (dd, H), 5.6 (t br, 1H), 6.449d, 1H), 6.48 (d, 1H), 7.58 (m, 6H), 7.92 (d, 4H).

Same procedure as compound 11. ¹H NMR of 12 (400 MHz, CDCl₃) δ 1.5 (s,9H), 2.83 (t, 2H), 3.62 (dt, 2H), 5.82 (t br, 1H), 6.4 (m, 2H), 7.18 (m,5H), 7.18 (m, 6H), 7.9 (m, 4H).

Method 2

By allowing a compound of formula (1) as defined above to react withpolymer bound compound of formula (14) wherein Z, R′ and R″ are definedas above in method 1.

These reactions may be carried out on functionalized cross linkedpolystyrene polymers such as Merrifield resin, Wang resin, Rink resin,Tentagel™ resin, in a solvent such as dimethylformamide (DMF),tetrahydrofuran (THF), or toluene, in the presence of a catalyst (e.g.Pd(OAc)₂, Pd(PPh₃)₄, Pd₂dba₃), a ligand (e.g. Ph₃P, Ph₃As, (o-tolyl)₃P)and a base (e.g. K₂CO₃, CsCO₃, Et₃N) at temperatures ranging from 23° C.to 130° C., for 1 to 60 hours.

EXAMPLES

For leading references see: a) Mathias (Synthesis 1979, 561). b)Sarantakis et al (Biochem. Biophys. Res. Commun. 1976, 73, 336). c)Hudson et al (Peptide Chemistry 1985 (Kiso, Y., ed.), 1986, ProteinResearch Foundation, Osaka.). d) Wang (J. Am. Chem. Soc. 1973, 95,1328). e) Lu et al (J. Org. Chem. 1981, 46, 3433.) e) Morphy et al(Tetrahedron Letters 1996, 37, 3209). e) Yedidia et al (Can. J. Chem.1980, 58, 1144).

To 10 g (11.2 mmol, 1 equiv) of Wang resin in 80 mL of drydichloromethane was added 33.6 mmol (3 equiv) of diisopropylcarbodiimideand the mixture was sonnicated under N₂ for 2 hours (final bathtemperature was 40° C.). Freshly distilled acrylic acid (33.6 mmol, 3equiv) and 4-dimethylaminopyridine (11.2 mmol, 1 equiv) were added andthe mixture was magnetically stirred for 16 hours at ambienttemperature. The resin was filtered and thoroughly washed withdichloromethne (500 mL), methanol (500 mL), dimethylformamide (500 mL),dichloromethane (500 mL) and methanol (500 mL) and dried in vacuo (0.1mmHg) for 24 hours. The coupling was repeated and resin 15 was filtered,washed and dried as above, and used directly in the next step.

To 8.2 g of acrylate Wang resin 15 was added 10.4 g (28.3 mmol) of4,4′-dibromobenzil, 437 mg of palladium (II) acetate (1.95 mmol), 1.25 gof tri-o-tolylphosphine (4.11 mmol), 95 mL of dimethylformamide followedby 3.3 mL (23.7 mmol) of triethylamine. The mixture was placed in a 100°C. preheated bath and stirred magnetically at 200 rpm for 2 hours. Theresin was filtered hot and washed thoroughly with hot dimethylformamide(500 mL), hot acetic acid (500 mL), methanol (500 mL), dichloromethane(500 mL), dimethylformamide (500 mL), dicliloromethane (500 mL) andmethanol (500 mL) and dried in vacuo (0.1 mmHg) for 24 hours. The linkerwas cleaved from the resin with a solution of 20% trifluoroacetic acidin dichloromethane for 20 min at ambient temperature. ¹H NMR formonobromo-monoacid linker (400 MHz, d₆-DMSO) δ 6.7 (d, 2H), 7.6 (d, 2H),7.8 (s, 4H), 7.9 (s, 4H).

To 10.2 g resin 16 was added 5.41 mL (37 mmol) of tert-butylacrylate,132 mg of palladium (II) acetate (0.592 mmol), 0.360 g oftri-o-tolylphosphirie (1.18 mmol), 31 mL of dimethylformamide followedby 1 mL (7.4 mmol) of triethylamine. The mixture was placed in a 100° C.preheated bath and stirred magnetically at 200rpm for 18 hours. Theresin was filtered hot and washed thoroughly with hot dimethylformamide(500 mL), hot acetic acid (500 mL), methanol (500 mL), dichloromethane(500 mL), dimethylformamide (500 mL), dichloromethe (500 mL) andmethanol (500 mL) and dried in vacuo (0.1 mmHg) for 24 hours. The linkerwas cleaved from the resin with a solution of 20% trifluoroacetic acidin dichloromethane for 20 min at ambient temperature. ¹H NMR for diacidlinker (400 MHz, d₆-DMSO) δ 6.7 (d, 2H), 7.6 (d, 2H), 7.9 (s, 8H).

To 1 g of acrylate resin 15 was added 1.02 g (2.8 mmol) ofmono-bromo-mono-tert-butylacrylate benzil (8), 0.044 g of palladium (II)acetate (0.19 mmol), 0.130 g of tri-o-tolylphosphine (0.41 mmol), 10 mLof dimethylformamide followed by a solution of 0.76 mL (5.7mmol) oftriethylamine in 10 mL of dimethylformamide. The mixture was placed in a100° C. preheated bath and stirred magnetically at 200 rpm for 2 hours .The resin was filtered hot and washed thoroughly with hotdimethylformamide (50 mL), water (50 mL), 10% sodium bicarbonate (50mL), 10% aqueous acetic acid (50 mL), water (50 mL), methanol (50 mL),dichloromethane (50 mL), methanol (50 mL), dichloromethane (50 mL) anddried in vacu (0.1 mmHg) for 24 hours. The linker was cleaved from theresin with a solution of 20% trifluoroacetic acid in dichloromethane for20 min at ambient temperature. ¹H NMR for diacid linker (400 MHz,d₆-DMSO) δ 6.7 (d, 2H), 7.6 (d, 2H), 7.9 (s, 8H).

Resin 18 was treated with a 1.0M solution of oxalyl chloride indichloromethane in the presence of a catalytic amount ofdimethylformamide for 1 hour and filtered. The resin was subsequentlytreated with a dichloromethane solution containing the alcohol (ROH),pyridine and 4-dimethylaminopyridine for 20 hours at 23° C. to yield themonoester resin 19.

Resin 18 was treated with a 1.0M solution of oxyalyl chloride indichloromethane in the presence of a catalytic amount ofdimethylformamide for 1 hour and filtered. The resin was subsequentlytreated with a dichloromethane solution containing the aromatic amine(ArN(R₁)H), pyridine and dimethylamiaopyridine for 20 hours at 23° C. toyield the mononmide resin 20.

Resin 18 was treated with a dichloromethane solution containing theamine (R₁R₂NH), EDCI and 4-dimethylaminopyridine for 20 hours at 23° C.to yield the monoamide resin 21.

General Methods for the Syntheses of Compounds (A2) and (A10)

Method 1

By allowing an aldehyde (R₁CHO) wherein R₁ is defined as above informula (A10) to react with itself.

These reactions may be carried out in a solvent or combination ofsolvents such as tetrahydrofuran (THF), dichlorometyhane (CH₂Cl₂), inthe presence of a catalyst (e.g. TiCl₃), and a base (e.g. pyridine) attemperatures ranging from −78° C. to 23° C., for 1 to 60 hours.

EXAMPLES

Prepared according to Araneo et al (Tetrahedron Lett. 1994, 35, 2213).The reaction was stirred for 4 hrs at 23° C. ¹H NMR of 22 (400 MHz,CDCl₃) δ 1.55 (s, 18H), 4.65 (s, 2H), 6.27 (d, 2H), 7.05 (d, 4H), 7.31(d, 4H), 7.5 (d, 2H).

¹H NMR of 23 (400 MHz, CD₃OD) δ 4.65 (s, 2H), 6.4 (d, 2H), 7.15 (d, 4H),7.4 (d, 4H), 7.6 (d, 2H). MS ESI (neg ion) for [M−H]⁻: 353 (calculated354).

Method 2

By allowing a compound of formula (A10)-1 as above to react with a acidchloride (R₂CO2H) and by subsequently oxidizing (A10)-2 wherein R₁ andR₂ are defined as in formula (A2).

The first step in this reaction may be carried out in a solvent such astetrahydrofuran (THF), dichloromethane (CH₂Cl₂), in the presence ofdiisopropyl carbodiimide (DIC) and a base (e.g. 4-dimethylaminopyridine)at temperatures ranging from 0° C. to 23° C., for 1 to 60 hours. Thesecond step in this reaction may be carried out in a solvent such asdichloromethane (CH₂Cl₂), in the presence of an oxidizing reagent (e.g.tetrapropylammonium perruthenate (VII) (TPAP)) and activated 4 Åmolecular sieves at temperatures ranging from 0° C. to 23° C., for 1 to60 hours.

EXAMPLES

To 50 mg of diol 22 (1 equiv) in 1 mL of dichloromethane was addeddiisopropyl carbodiimide (0.4 equiv) and the reaction was stirred for 1hour at 23° C. To the solution was added 4-dimethylaminopyridine (0.1equiv) followed by para-methoxybenzoic acid (0.4 equiv) in 5 mL oftetrahydrofuran and the mixture was stirred for an additional 3 hours at23° C. The reaction was diluted with ethyl acetate and washed with 1NHCl, saturated sodium bicarbonate and the organic layer was dried oversodium sulfate. The crude mixture was separated using radialchromatography (ethyl acetate-hexane eluent). ¹H NMR of 24 (400 MHz,CDCl₃) δ 1.55 (s, 18H), 3.8 (s, 3H), 5.05 (d, 1H), 6.0 (d, 1H), 6.25 (d,1H), 6.3 (d, 1H), 6.9 (d, 2H), 7.1 (d, 4H), 7.32 (m, 4H), 7.45 (d, 1H),7.48 (d, 1H), 8.0 (d, 1H).

¹H NMR of 25 (400 MHz, CD₃OD) δ 3.82 (s, 3H), 5.08 (d, 1H), 6.02 (d,1H), 6.4 (d, 2H), 6.9 (d, 2H), 7.22 (d, 4H), 7.42 (d, 4H), 7.6 (d, 2H),8.03 (d, 2H). MS ESI (neg ion) for [M−H]⁻: 487 (calculated 488).

Hydroxyester 24 (1 equiv) was oxidized to ketoester 26 at 23° C. inCH₂Cl₂, in the presence of catalytic amount of TPAP (0.1 equiv),N-methylnorpholine oxide (2 equiv) and 4 Å activated powdered molecularsieves (500 mg/mol of substrate). ¹H NMR of 26 (400 MHz, CDCl₃) δ 1.55(s, 18H), 3.8 (s, 3H), 6.25 (d, 1H), 6.29 (d, 1H), 6.9 (d, 2H), 7.0 (s,1H), 7.5 (m, 10H), 7.95 (d, 1H), 8.02 (d, 1H).

¹H NMR of 27 (400 MHz, CD₃OD) δ 3.82 (s, 3H), 6.45 (d, 1H), 6.55 (d,1H), 6.95 (d, 2H), 7.18 (s, 1H), 7.65 (m, 10H), 8.0 (d, 1H), 8.08 (d,1H).

Method 3

By allowing an acid chloride (R₁COCl) to react with an aldehyde (R₂CHO)wherein R₁, R₂ are defined as above in formula (A2) and by subsequentlyoxidizing (A10)-3.

The first step in this reaction may be carried out in a solvent or acombination of solvents such as tetrahydrofuran (THF), dichloromethane(CH₂Cl₂), in the presence of a catalyst (e.g. TiCl₃), and a base (e.g.pyridine) at temperatures ranging from −78° C. to 23° C., for 1 to 60hours. The second step in this reaction may be carried out in a solventsuch as dichloromethane (CH₂Cl₂), in the presence of an oxidizingreagent (e.g. tetrapropylammonium perruthenate (VII) (TPAP)) andactivated 4 Å molecular sieves at temperatures ranging from 0° C. to 23°C., for 1 to 60 hours.

EXAMPLES

Prepared according to Araneo et al (Tetrahedron Lett. 1994, 35, 2213).The reaction was stirred for 4 hrs at 23° C. the crude mixture wasseparated by flash chromatography (ethyl acetate in hexane eluent) toyield hydroxyester 28. ¹H NMR of 28 (400 MHz, CDCl₃) δ 1.55 (s, 18H),1.6 (m, 4H), 2.2-2.4 (m, 4H), 3.6 (s, 3H), 4.9 (d, 1H), 5.85 (d, 1H),6.25 (d, 1H), 6.3 (d, 1H), 7.07 (m, 4H), 7.3 (m, 4H), 7.45 (m, 2H).

¹H NMR of 29 (400 MHz, CD₃OD) δ 1.5 (m, 4H), 2.3 (m, 2H), 2.4 (m, 2H),3.6 (s, 3H), 4.95 (d, 1H), 5.85 (d, 1H), 6.4 (d, 2H), 7.2 (m, 4H), 7.42(d, 4H), 7.6 (d, 2H). MS ESI (neg ion) for [M−H]⁻: 495 (calculated 496).

Hydroxyester 28 was oxidized to ketoester 30 as above. ¹H NMR of 30 (400MHz, CDCl₃) δ 1.55 (s, 18H), 1.65 (s br, 4H), 2.3 (m, 2H), 2.5 (m, 2H),3.6 (s, 3H), 6.3 (d, 1H), 6.35 (d, 1H), 6.78 (s, 1H), 7.4-7.6 (m, 8H),7.9 (d, 2H).

General Method for the Synthesis of Compounds (A3)

Method 1

By allowing a carboxylic acid (R₁CO₂H) to react with an isocyanide(R₂NC) and an aldehyde (R₃CHO) wherein R₁, R₂, and R₃ are defined asabove in formula (A3).

These reactions may be carried out in a solvent or a combination ofsolvents such as dichloromethane (CH₂Cl₂), chloroform (CHCl₃), methanol(MeOH), tetrahydrofuran (THF) or acetonitrile (CH₃CN), in the presenceor absence of a catalyst (e.g. ZnCl₂, MgBr₂) at temperatures rangingfrom −78° C. to 80° C., for 1 to 60 hours.

EXAMPLES

Prepared according to Passerini (Gazz. Chim. Ital. 1926, 56, 826).

A solution of the carboxylic acid, aldehyde and isocyanide in a givensolvent selected from tetrahydrofuran, acetonitrile, ethyl ether orchloroform was stirred between 0° and 25° C. for 1 to 3 days. Thesolution was diluted with ethyl acetate, washed with saturated sodiumbicarbonate and dried over sodium sulfate. The solvent was removed invacuo and the residue was purified by silica gel chromatography.

¹H NMR of 32(400 MHz, d₆-acetone) δ 0.8 (t, 3H), 1.1-1.6 (m, 9H), 1.97(m, 1H), 3.9 (m, 2H), 4.1 (m, 2H), 5.3 (t, 1H), 6.62 (d, 1H), 7.7 (d,1H), 7.8 (d, 2H), 8.05 (d, 2H).

Method 2

By allowing a carboxylic acid (R₁CO₂H) and an aldehyde (R₃CHO) to reactwith a polymer bound isocyanide (R₂NC) wherein R₁, R₂, and R₃ aredefined as above in formula (A3).

These reactions may be carried out on functionalized cross linkedpolystyrene polymers such as Merrifield resin, Wang resin, Rink resin,Tentagel™ resin, in a solvent or a combination of solvents such asdichloromethane (CH₂Cl₂), chloroform (CHCl₃), methanol (MeOH),setrahydrofuran (THF), acetonitrile (CH₃CN), in the presence or absenceof a catalyst (e.g. ZnCl₂, MgBr2) at temperatures ranging from −78° C.to 80° C., for 1 to 60 hours. The product maybe released from thepolymer by conditions known to those skilled in the art.

EXAMPLES

Prepared according to Zhang et al (Tetrahedron Letters 1996, 37, 751).

A solution of the carboxylic acid 3 in tetrahydrofuran was added to amixture of the aldehyde and isocyanide resin 33 in tetraydrofuran oracetonitrile. The mixture was stirred at 25° C. or 60° C. for 1 to 3days. The resin was filtered and washed with dichloromethane andmethanol and dried. Compounds 34 were isolated after treatment of theresin with a solution of 50% trifluoroacetic acid in dichloromethane for1 hour at 23° C. and removal of the solvent in vacuo.

A solution of the carboxylic acid in tetrahydrofuran was added to amixture of the aldehyde 4 and isocyanide resin 33 in tetrahydrofuran oracetonitrile. The mixture was stirred at 25° C. or 60° C. for 1 to 3days. The resin was filtered and washed with dichloromethane andmethanol and dried. Compounds 35 were isolated after treatment of theresin with a solution of 50% trifluoroacetic acid in dichloromethane for1 hour at 23° C. and removal of the solvent in vacuo.

TABLE 1

MWt [M − H]⁻ Compound R₂ n (Calculated) (Found) 36

2 475; 477 474; 476 37

5 519 518 38 C₆H₁₄CHO 2 405 404 39 C₆H₁₄CHO 5 447 446 40 C₉H₂₀CHO 2 447446 41 C₉H₂₀CHO 5 489 488

TABLE 2

MWt [M − H]⁻ Compound R₁ n (Calculated) (Found) 42

2 427 426 43

5 469 468 44

2 442 441 45

2 441 440 46

5 483 482 47

2 419 418 48

5 519 518 49

2 417 416 50

5 459 458

General Method for the Synthesis of Compounds (A4)

By allowing an acid chloride (R₁COCl) to react with an aldehyde (R₂CHO)wherein R₁, R₂ are deined as above in formula (A4)

These reactions may be carried out in a solvent or combination ofsolvents such as tetrahydrofuran (THF), dichloromethane (CH₂Cl₂), in thepresence of a catalyst (e.g. TiCl₃), and a base (e.g. pyridine) attemperatures ranging from −78° C. to 23° C., for 1 to 60 hours.

EXAMPLE

Prepared according to Aranco et al (Tetrahedron Lett. 1994, 35, 2213).¹H NMR of 51 (400 MHz, CDCl₃) δ 1.45 (s, 9H), 1.5 (m, 4H), 2.1-2.3 (m,4H), 3.6 (s, 3H), 4.6 (s, 1H), 6.25 (d, 1H), 6.97 (d, 2H), 7.25 (d, 2H),7.5 (d, 1H).

General Methods for the Sythesis of Compounds (A6)

Method 1

By allowing a compound of formula (A5) to react with an aldehydi(R₂CHO), a primary amine (R₁NH₂) and ammonium acetate wherein R₁, R₂, R₃and R₄ are defined as above in formula (A6).

These reactions may be carried out in a solvent such as acetic acid(AcOH) at temperatures ranging from 23° C. to 120° C., for 1 to 60 hours.

EXAMPLES

Prepared according to Krieg et al (Z Naturforsch teil 1967, 22b, 132).

To 47 mg of 6 (0.1mmol, 1.0 equiv), R₂CHO (0.1 mmol, 1.0 equiv) in 1 mLof acetic acid was added 231 mg of ammoniumn acetate (3.0 mmol, 30equiv) in 0.5 mL of acetic acid and the mixture was placed in 100° C.preheated oil bath for 1 hour. The solution was then poured into etherand washed with saturated sodium bicarbonate.

The organic layer was dried over sodium sulfate, filtered andconcentated in vacuo to yield the desired imidazoles 52 which werepurified by preparative thin layer chromatography with ethylacetate-hexane or methanol-dichloromethane as eluent

TABLE 3

MWt. MWt. Entry R₂ (Calc.) (Obs.) 54

480 479 55 H —* —* 56

525 526 57

481 482 58

—* —* 59

511 512 60

—* —* 61

—* —* 62

521 522 63

487 488 64

533 534 65

—* —* 66

—* —* 67

—* —* 68

—* —* 69

482 483 70

442 441 71

494 495 72

526 527 73

—* —* 74

489 490 75 Ph 436 435 76

—* —* 77 n-C₅H₁₁ —* —* 78

454 455 79

—* —* 80

560 561 81

536 537 82

—* —* 83

—* —* 84

526 527 85

480 481 86

494 495 87

542 543 88

561 562 89

476 477 90

493 494 *“—” data not available.

54 (400 MHz, CDCl₃-CD₃OD 10:1) δ 6.23 (d, 2H), 7.3-7.48 (m, 10H), 7.88(d, 2H), 8.02 (d, 2H).

55 (400 MHz, CD₃OD) δ 6.5 (d, 2H), 7.52 (d, 4H), 7.7 (m, 6H), 9.1 (s,1H).

56 (400 MHz, CD₃OD) δ 6.3 (s, 2H), 6.52 (d, 2H), 7.4-7.9 (m, 12H).

57 (400 MHz, CD₃OD) δ 6.52 (d, 2H), 7.50-8.36 (m14H).

58 (400 MHz, CDCl₃-CD₃OD 10:1) δ 3.7 (s, 3H), 6.3 (d, 2H), 6.85 (d, 2H),7.4 (m, 8H), 7.5 (d, 2H), 7.85 (d, 2H).

59 (400 MHz, CD₃OD) δ 3.98 (s, 3H), 6.52 (d, 2H), 7.50-7.76 (m, 13H).

60 (400 MHz, CDCl₃-CD₃OD 10:1) δ 6.3 (d, 2H), 6.5 (br s, 1H), 6.85 (d,2H), 7.3-7.6 (m, 12H).

61 di-tert-butyl ester (400 MHz, CDCl₃-CD₃OD 6:1) δ 1.4 (s, 18H), 6.2(d, 2H), 6.9 (t, 1H), 7.2-7.42 (m, 11H), 7.58 (d, 1H), 7.62 (d, 1H).

62 (400 MHz, CD₃OD) δ 6.50 (d, 2H), 7.30-8.70 (m, 12H).

63 (400 MHz, CD₃OD) δ 6.54 (d, 2H), 7.46-8.60 (n, 16H).

64 (400 MHz, CD₃OD) δ 3.90 (s, 3H), 6.50 (d, 2H), 7.50-8.30 (m, 13H).

65 di-tert-butyl ester (400 MHz, CDCl₃-CD₃OD 6:1) δ 1.4 (s, 18H), 6.2(d, 2H), 6.65 (t, 1H), 7.3-7.5 (m, 12H).

66 di-tert-butyl ester (400 MHz, CDCl₃-CD₃OD 6:1) δ 1.4 (s, 18H), 6.2(dd, 2H), 7.1 (q, 1H), 7.3-7.5 (m, 10H), 7.6 (br d, 1H), 7.7 (dd, 1H).

67 di-tert-butyl ester (400 MHz, CDCl₃-CD₃OD 6:1) δ 1.4 (s, 18H), 6.2(d, 2H), 7.1 (t, 2H), 7.3-7.5 (m, 10H), 7.75 (m, 1H).

68 (400 MHz, CD₃OD) δ 1.2 (t, 6H), 3.48 (q, 4H), 6.52 (d, 2H), 7.3-8.02(m, 15H).

69 (400 MHz, CD₃OD) δ 3.96 (s, 3H), 6.52 (d, 2H), 7.04-7.70 (m, 13H).

70 (400 MHz, CD₃OD) δ 6.46 (d, 2H), 7.14 (d, 1H), 7.50-7.8C (m, 12H).

71 (400 MHz, CD₃OD) δ 4.32 (m, 4H), 6.52 (d, 2H), 7.1-7.7 (m, 13H).

72 (400 MHz, CD₃OD) δ 3.90-4.02 (3s, 9H), 6.50 (d, 2H), 6.90-7.80 (m,12H).

73 (400 MHz, CD₃OD) δ 6.5 (d, 2H), 7.55 (d, 4H), 7.65 (m, 6H), 7.95 (d,2H), 8.15 (d, 2H).

74 (400 MHz, CD₃OD) δ 3.95 (s, 3H), 6.56 (d, 2H), 6.9-7.82 (m, 14H).

75 (400 MHz, CDCl₃-CD₃OD 10:1) δ 6.34 (d, 2H), 7.3-7.4 (m, 11H), 7.52(d, 2H), 8.92 (d, 2H).

76 di-tert-butyl ester (400 MHz, CDCl₃-CD₃OD 6:1) δ 1.4 (s, 18H), 6.2(br d, 2H), 6.9 (m, 1H), 7.05 (m, 1H), 7.3-7.5 (m, 10H).

77 (40 MHz, CDCl₃-CD₃OD 6:1) δ 0.9 (m, 5H), 1.3 (m, 2H), 1.7 (m, 2H),2.9 (t, 2H), 6.35 (d, 2H), 7.3-7.6 (m, 10H).

78 (400 MHz, CD₃OD) δ 6.50 (d, 2H), 7.40-7.9 (m 14H).

79 di-tert-butyl ester (400 MHz, CDCl₃) δ 1.4 (s, 18H), 6.3 (d, 2H), 7.1(d, 2H), 7.22 (d, 1H), 7.34 (t, 2H), 7.4-7.7 (m, 14H), 7.9 (d, 2H).

80 (400 MHz, CD₃OD) δ 6.54 (d, 2H), 7.6-8.0 (m, 19H).

81 (400 MHz, CD₃OD) δ 6.54 (d, 2H), 7.6-8.90 (m, 19H).

82 (400 MHz, CD₃OD) δ 6.50 (d, 2H), 7.58-8.0 (m, 14H).

83 (400 MHz, CD₃OD) δ 3.96 (s, 3H), 6.52 (d, 2H), 7.36-7.90 (m, 13H).

84 (400 MHz, CD₃OD) δ 6.50 (d, 2H), 7.55-7.70 (m, 10H).

85 (400 MHz, CD₃OD) δ 6.12 (s, 2H), 6.56 (d, 2H), 7.10-7.60 (m, 13H).

86 (400 MHz, CD₃OD) δ 2.20 (s, 3H), 2.40 (s, 3H), 3.90 (s, 3H), 6.52 (d,2H), 7.10-7.70 (m, 12H).

87 (400 MHz, CD₃OD) δ 5.20 (s, 2H), 6.56 (d, 2H), 7.22-7.98 (m, 19H).

88 (400 MHz, CD₃OD) δ 1.52 (2s, 12H), 1.74 (s, 4H), 2.42 (s 3H), 6.52(d, 2H), 7.40-7.68 (m, 13H).

89 (400 MHz, CD₃OD) δ 1.12 (t, 2H), 3.0 (m, 4H), 6.56 (d, 2H), 7.52-7.62(m, 13H).

90 (400 MHz, CD₃OD) δ 2.14 (s, 3H), 6.54 (d, 2H), 7.58-8.0 (m, 14H).

Prepared according to Krieg et al (Z Naturforsch teil 1967, 22b, 132).

¹H NMR of 91 (400 MHz, CD₃OD) δ 3.9 (s, 3H), 6.2 (d, 2H), 6.95 (d, 2H),7.2 (d, 2H), 7.4-7.6 (m, 6H), 7.9 (d, 2H).

Prepared according to Krieg et al (Z Naturforsch teil 1967, 22b, 132).

TABLE 4

MWt MWt Entry R₁ R₂ (Calc.) (Obs.) 94 n-C₄H₉ H 416 415 95 n-C₄H₉

498 497 96 Ph

518 517 97 n-C₄H₉

522 521 98 Ph

542 541 99 Ph H 436 435 100 

582 581

94 (400 MHz, CD₃OD) δ 0.8 (t, 3H), 1.22 (m, 2H), 1.62 (m, 2H), 4.10 (t,2H), 6.42 (d, 1H), 6.58 (d, 1H), 7.32-7.80 (m, 10H), 9.18 (s, 1H).

95 (400 MHz, CD3OD) δ 0.64 (t, 3H), 1.04 (m, 2H), 1.58 (m, 2H), 4.20 (t,2H), 6.42 (d, 1H), 6.62 (d, 1H), 7.42-8.0 (m, 13H).

96 (400 MHz, CD3OD) δ 6.42 (2d, 2H), 7.12-7.68 (m, 18H).

97 (400 MHz, CD₃OD) δ 0.6 (t, 3H), 1.0 (m, 2H), 1.38 (m, 2H), 4.12 (t,2H), 3.84 (s, 3H), 6.42 (d, 1H), 6.62 (d, 1H), 7.22-7.8 (m, 13H).

98 (400 MHz, CD₃OD) δ 3.80 (s, 3H), 6.44 (2d, 2H), 6.94-7.68 (m, 19H).

99 (400 MHz, CD₃OD) δ 6.44 (2d, 2H), 7.20-7.60 (m, 15H), 9.2 (s, 1H).

100 (400 MHz, CD₃OD) δ 1.22 (s, 9H), 2.40 (s, 3H), 6.36-6.44 (2d, 2H),7.26-7.60 (m, 18H).

Method 2

By allowing a polymer bound compound of formula (A5)-2 to react with analdehyde (R₂CHO), A priary amine (R₁NH₂) and ammonium acetate whereinR₁, R₂, R₃ and R₄ are fined as above in formula (A6).

These reactions may be carried out on functionalized cross linkedpolystyrene polymers such as Merrifield resin, Wang resin, Rink resin,Tentagel™ resin, in a solvent such as acetic acid (AcOH) at temperaturesranging from 23° C. to 120° C., for 1 to 60 hours. The product may bereleased from the polymer using conditions known to those skilled in theart.

EXAMPLES

To resin 17 were added excess NH₄OAc and R₂CHO and acetic acid and themixture was heated at 100° C. for 15 hours, cooled to 23° C. and washedwith methanol and dichloromethane and dried under vacuum. Thetrifluoroacetate salts of imidazoles 101 were isolated followingtreatment of the resin with a solution of 20% trifluoroacctic acid indichloromethane for 20 minutes at 23° C.

Same procedure as imidazoles 101.

TABLE 5 MWt. MWt. Entry R₁ R₂ (Calc.) (Obs.) 103 Me

495 496 104

620 621 105

602 603 106

586 587 107

638 639 108

634 635 109 2-propyl

496 497 110 2-propyl

523 524 111 2-propyl

553 554 112 2-indanyl

627 628 113

626 627

Same procedure as imidazoles 101.

Same procedure as imidazoles 101.

TABLE 6

MWt. MWt. Entry R₁ R₂ (Calc.) (Obs.) 116

559 560 117

562 563 118

633 634 119

642 643 120

592 593 121

579 580 122 n-propyl

508 509 123 n-propyl

562, 564 563, 565 124 n-butyl

528 529 125 n-heptyl

579 580 126 n-octyl

566 567 127 n-octyl

619 620 128

612 613

Method 3

By allowing a compound of formula (129) (J. Org. Chem., 1995, 60, 8231;J. Org. Chem., 1993, 58, 4785) to react with an aldehyde (R₂CHO), aprimary amine (R₁NH₂) and ammonium acetate wherein R₁, R₂, R₃ and R₄ aredefined as above in formula (A6).

These reactions may be carried out in a solvent such as acetic acid(AcOH) at temperatures ranging from 23° C. to 120° C., for 1 to 60hours.

EXAMPLES

Prepared according to Wasserman et al (J. Org. Chem., 1995, 60, 8231; J.Org. Chem., 1993, 58,4785). Benzyl (triphenylphosphoranylidene) acetate(130) was purchased from Aldrich chemical company and used directly. ¹HNMR of 131 (400 MHz, CDCl₃) δ 1.5 (s, 9H), 4.62 (s, 2H), 6.3 (d, 1H),6.62 (d, 2H), 7.0 (t, 2H), 7.1 (t, 1H), 7.38-7.8 (m, 20H). TLC:R_(ƒ)=0.5 (30% ethyl acetate-hexane).

Prepared according to Wasserman et al (J. Org. Chem., 1995, 60, 8231; J.Org. Chem., 1993, 58 785). ¹H NMR of 132 (400 MHz, CDCl₃) δ 1.5 (s, 9H),5.1 (s, 2H), 5.15 (br s, 2H, 2×H—O), 6.4 (d, 1H), 6.95 (d, 2H), 7.1 (t,2H), 7.18 (t, 1H), (7.4 (d, 2H), 7.5 (d, 1H), 7.9 (d, 2H). TLC:R_(ƒ)=0.7 (30% ethyl acetate-hexane).

¹H NMR of 133 (400 MHz, CDCl₃-CD₃OD, 8:1) δ 5 (s, 2H), 6.4 (d 1H),6.9-7.16 (m, 5H), 7.35 (d, 2H), 7.53 (d, 1H), 7.9 (d, 2H).

Prepared according to Brackeen et al (Tetrahedron Letters 1994, 35,1635). For other approaches to imidazole-4-carboxylates see: a) Nunamiet al (J. Org. Chem. 1994, 59, 7633). b) Heindel et al (TetrahedronLetters 1971, 1439). ¹H NMR of 134 (400 MHz, 8:1 CDCl₃-CD₃OD) δ 5.2 (s,2H), 6.4 (d, 1H), 7.25 (br s, 5H), 7.5 (d, 2H), 7.6 (d, 1H), 7.7 (d,2H), 8.3 (s, 1H).

Method 4

By allowing a compound of formula (129) to react with a polymer boundaldehyde (R₁CHO), a primay amine (R₂NH₂) and ammonium acetate whereinR₁, R₂, R₃ and R₄ are defined as above in formula (A6).

This reaction may be carried out on functionalized cross linkedpolystyrene polymers such as Merrifield resin, Wang resin, Rink resin,Tentage™ resin, in a solvent such as acetic acid (AcOH) at temperaturesranging from 23° C. to 120° C., for 1 to 60 hours. The product maybereleased from the polymer using conditions known to those skilled in theart.

EXAMPLES

For leading references see: a) Mathias (Synthesis, 1979, 561). b)Sarantakis et al (Biochem. Biophys. Res. Commun. 1976, 73, 336). c)Hudson et al (Peptide Chemistry 1985 (Kiso, Y., ed.), 1986, ProteinResearch Foundation, Osaka.). d) Wang (J. Am. Chem. Soc. 1973, 95,1328). e) Lu et al (J. Org. Chem. 1981, 46, 3433). To 6 mmol (1 equiv)of Wang resin in 130 mL of dry dimethylformamide was added 18 mmol (3equiv) of diisopropylcarbodiimide and the mixture was sonnicated for 4hours (final bath temperature was 37° C.). 4-Formylcinnamic acid (18mmol, 3 equiv) and 4-dimethylaminopyridine (6 mmol, 1 equiv) were addedand the mixture was magnetically stirred for 48 hours at ambienttemperature. The resin was filtered and thoroughly washed withdimethylforamide (500 mL), methanol (500 mL), dichloromethane (500 mL)and methanol (500 mL) and dried in vacuo (0.1 mmHg) for 24 hours. Acoupling yield of 80% was established by cleaving 100 mg of the resinwith a solution of 20% trifluoroacetic acid in dichloromethane for 20min at ambient temperature.

To 60 mg (0.048 mmol, 1,0 equiv) of 135 was added 40 mg (0.097 mmol, 2.0equiv) of 132 followed by 37 mg (0.481 mmol, 5.0 equiv) of anunoniumacetate and 0.2 mL of acetic acid. The mixture was heated to 100° C. for15 hours, filtered, washed with dimethylformanide, dichloromethane,methanol and dichloromethane. The crude product was isolated bytreatment of the polymer with a solutioa of 50% trifuoroacetic acid indichloromethane for 1 hour at 23° C. The solvent was removed and theresidue was purified by prepatitive thin layer chromatography (20%methanol-dichloromethane eluent). ¹H NMR of 136 (400 MHz, CD₃OD) δ 5.15(s, 2H), 6.48 (d, 1H), 6.55 (d, 1H), 7.25 (br s, 4H), 7.5-7.8 (m, 9H),8.1 (d, 1H). MS (ESI negative ion) [M−H]⁻: 493;

Method 5

By allowing a primary amine (R₁NH₂), a carboxylic acid (R₂CO₂H) and aketoaldehyde (R₄COCHO) to react with a polymer bound isocyanide (R₃NC)and by subsequently cyclizing compound 137 with ammonium acetate whereinR₁, R₂, R₃ and R₄ are defined as above in formula (A6).

The first step in this reaction may be carried out on functionalizedcross linked polystyrene resins such as Merrifield resin, Wang resin,Rink resin, Tentage™ resin, in a solvent or a combination of solventssuch as dichloromethane (CH₂Cl₂), chloroform (CHCl₃), methanol (MeOH),tetrahydrofuran (THF) or acetonitrile (CH₃CN), in the presence orabsence of a catalyst (e.g. ZnCl₂, MgBr₂) at temperatures ranging from−78° C. to 80° C., for 1 to 60 hours. The second step in this reactionmay be carried out in a solvent such as acetic acid (AcOH) attemperatures ranging from 23° C. to 120° C., for 1 to 60 hours.

Prepared according to Gunn et al (J. Org. Chem. 1977, 42, 754).

Prepared according to Zhang et al (Tetrahedron Letters 1996, 37, 751).

Prepared according to Zhang et al (Tetrahedron Letters 1996, 37, 751).

¹H NMR of mono tert-butyl ester of 140 (400 MHz, CDCl₃) δ 1.1 (m, 2H),1.2 (d, 3H), 1.3 (m, 2H), 1.5 (s, 9H), 1.56 (m, 2H), 2.2 (m, 2H), 2.9(m, 1H), 3.1 (m, 1H), 3.2 (m, 1H), 3.8 (s, 3H), 4.6 (m, 2H), 6.1 (d,1H), 6.9 (t, 4H), 7.1 (m, 5H), 7.4 (d, 2H), 7.6 (d, 1H).

Method 6

By allowing a carboxylic acid (R₁CO₂H) to react with an isocyanide(R₃NC) and a ketoaldehyde (R₂COCHO) and by allowing compound 141 tocyclize in the presence of ammonium acetate, wherein R₁, R₂, and R₃ aredefined as above in formula (A6).

The first step in this reaction reaction may be carried out in a solventor a combination of solvents such as dichloromethane (CH₂Cl₂),chloroform (CHCl₃), methanol (MeOH), tetrahydrofuran (THF), acetonitrile(CH₃CN), in the presence or absence of a catalyst (e.g. ZnCl₂, MgBr₂) attempratures ranging from −78° C. to 80° C., for 1 to 60 hours. Thesecond step in this reaction may be carried out in a solvent such asacetic acid (AcOH) at temperatures ranging from 23° C. to 120° C., for 1to 60 hours.

EXAMPLES

Prepared according to Bossio et al (Liebigs Ann. Chem. 1991, 1107).

To an ethyl ether mixtue of the carboxylic acid and ketoaldehyde at 0°C. was added dropwise an ethyl ether solution of the isocyanide. Themixture was warmed to 25° C. and stirred for 2 hours to 3 days. Thesolution was diluted with ethyl acetate, washed with saturated sodiumbicarbonate and dried over sodium sulfate. The solvent was removed invacuo and the residue was purified by silica gel chromatography to yieldα-Acyloxy-β-ketoamide 142.

A solution of the α-Acyloxy-β-ketoamide 142 (1 equiv) and ammoniunacetate(30 equiv) in acetic acid was heated at 100° C. for 2 to 15hours. The reaction was cooled to 23° C., diluted with ethyl acetate,washed with saturated sodium bicarbonate and dried over sodium sulfate.Solvent was removed in vacuo and the crude mixture was sepaprated bysilica gel chromatography to provide imidazole 143.

¹H NMR of 144 (400 MHz, d₆-acetone) δ 0.85 (t, 3H), 1.2-1.6 (m, 4H), 3.3(m, 2H), 6.55 (s, 1H) 6.62 (d, 1H), 7.3 (t, 2H), 7.7 (d, 1H), 7.82 (d,2H), 8.1 (d, 2H), 8.25 (dd, 2H).

¹H NMR of 146 (400 MHz, d₆-acetone) δ 0.9 (t, 3H), 1.4 (m, 2H), 1.6 (m,2H), 3.35 (m, 2H), 6.58 (d, 1H), 7.12 (t, 2H), 7.65 (d, 1H), 7.78 (d,2H), 8.1 (s br, 1H), 8.05 (m, 1H), 8.2 (d, 2H).

General Method for the Synthesis of Compounds (A7)

By allowing a carboxylic acid (R₁CO₂H) to react with an isocyanide(R₃NC) and a ketoaldehyde (R₂COCHO) wherein R₁, R₂, and R₃ are definedas above in formula (A7) and by allowing compound 141 to cyclize in thepresence of ammonium acetate.

The first step in this reaction reaction may be caried out in a solventor a a, combination of solvents such as dichloromethac (CH₂Cl₂),chloroform (CHCl₃), methanol (MeOH), tetaydrofuran (THF), acetonitrile(CH₃CN), in the presence or absence of a catalyst (e.g. ZnCl₂, MgBr₂) attemperatures ranging from −78° C. to 80° C., for 1 to 60 hours. Thesecond step in this reaction may be carried out in a solvent such asacetic acid (AcOH) at temperatures ranging from 23° C. to 120° C., for 1to 60 hours.

EXAMPLES

Prepared according to Bossio et al (Liebigs Ann. Chem. 1991, 1107).

A solution of the α-Acyloxy-β-ketoamide 141(1 equiv) and ammoniumacetate (2 equiv) in acetic acid was heated at 100° C. for 2 to 15hours. The reaction was cooled to 23° C., diluted with ethyl acetate,washed with saturated sodium bicarbonate and dried over sodium sulfate.Solvent was removed in vacuo and the crude oxazole 147 was purified bysilica gel chromatography.

¹H NMR of 148 (400 MHz, d₆-acetone) δ 0.9 (t, 3H), 1.4 (m, 2H), 1.6 (m,2H), 3.42 (m, 2H), 6.63 (d, 1H), 7.2 (t, 2H), 7.7 (d, 1H), 7.9 (d, 2H),8.18 (s br, 1H), 8.25 (d, 2H), 8.6 (m, 1H).

General Methods for the Synthesis of Compounds (A8) and (A9)

Method 1

By allowing a compound of formula (A5) to react with compound of formula(149) wherein R₁, R₂, R₃, R₄, R₅, and R₆ are defined as above in formula(A8).

These reactions may be carried out in a solvent or a combination ofsolvents such as dioxane or acetic acid (AcOH) at temperatures rangingfrom 23° C. to 120° C., for 1 to 60 hours.

EXAMPLES

A solution of 0.1 mmol of diamine 150 and 0.1 mmol of 6 in 1.2 mL of1,4-dioxane-acetic acid (5:1) was heated at 100° C. Upon completion ofthe reaction as judged by thin layer chromatography, ethyl acetate wasadded and the organic layer was washed with water, 0.5M citric acid, 10%sodium bicarbonate and dried over sodium sulfate. The compounds werepurified using silica gel chromatography.

TABLE 7

Compound R₁ R₂ R₃ R₄ 152 H H NO₂ H 153 H Cl Cl H 154 H H CH₃ H 155 H HCO₂H H 156 H H CO₂Me H 157 H H H H

152 (400 MHz, CD₃OD) δ 6.5 (d, 2H), 7.3 (s, 1H), 7.4-7.8 (m, 10H), 7.9(s, 1H), 8.05 (d, 1H). MS ESI (pos ion) for [M+H]⁺: 468 (calculaued467).

153 (400 MHz, CD₃OD) δ 6.48 (d, 2H), 7.5 (dd, 8H), 7.6 (d, 2H), 8.24 (s,2H). MS ESI (pos ion) for [M+H]⁺: 491, 492 (calculated 490, 491).

154 (400 MHz, CD₃OD) δ 3.3 (s, 3H), 6.5 (d, 2H), 7.59 (s, 8H), 7.62 (d,2H), 8.3 (d, 1H), 8.55 (d, 1H), 8.95 (s, 1H). MS ESI (pos ion) for[M+H]⁺: 437 (calculated 436).

155 (400 MHz, d₆-DMSO) δ 6.56 (d, 2H), 7.5 (m, 6H), 7.65 (d, 4H), 8.2(d, 1H), 8.3 (d, 1H) 8.6 (s, 1H). MS ESI (neg ion) for [M−H]⁻: 465(calculated 466).

156 (400 MHz, CD₃OD) δ 6.56 (d, 2H), 7.5 (s br, 8H), 7.65 (d, 2H), 8.2(d, 1H), 8.3 (d, 1H), 8.7 (s, 1H). MS ESI (neg ion) for [M−H]⁻: 479(calculated 480).

157 (400 MHz, d₆-DMSO) δ 6.52 (d, 2H), 7.54-8.16 (m, 14H).

Method 2

By allowing a compound of formula (A5) to react with compound of formula(158) wherein R₁, R₂, R₃, R₄, R₅ are defined as above in fonmula (A9).

These reactions may be carried out in a solvent or a combination ofsolvents such as dioxane or acetic acid (AcOH) at temperatures rangingfrom 23° C. to 120° C., for 1 to 60 hours.

EXAMPLES

¹H NMR of 160 (400 MHz, CD₃OD) δ 6.5 (d, 2H), 7.5-7.7 (m, 12H), 7.95 (m,1H), 8.65 (d, 1H), 9.15 (s, 1H). MS ESI (pos ion) for [M+H]⁺: 424(calculated 423).

General Method for the Synthesis of Compounds (A10)

By allowing a compound of fornula (A10)-1 precpaid as above to reactwith a carboxylic acid (R₂CO₂H) wherein R₁ and R₂ are defined as abovein formula (A10).

These reactions may be carried out in a solvent such as tetrahydrofuran(THF), dichloromethane (CH₂Cl₂), in the prence of diisopropylcarbodiimide (DIC) and a base (e.g. 4,4-dimethylaminopyridine) attemperatures ranging from 0° C. to 23° C., for 1 to 60 hours.

EXAMPLES

To 50 mg of diol 22 in 1 mL of dichloromethane was added diisopropylcarbodiimide (2.2 equiv) and the reaction was stirred for 1 hour at 23°C. To the solution was added 4,4-dimethylaminopyridine (0.2 equiv)followed by para-methoxybenzoic acid (2.2 equiv) in 5 mL oftetrahydrofuran and the mixture was stirred for an additional 3 hours at23° C. The reaction was diluted with ethyl acetate and washed with 1NHCl, saturated sodium bicarbonate and the organic layer was dried oversodium sulfate. The crude mixture was purified using radialchromatography (ethyl acetate-hexane eluent). ¹H NMR of 161 (400 MHz,CDCl₃) δ 1.5 (s, 18H), 3.8 (s, 6H), 6.25 (d, 2H), 6.32 (s, 2H), 6.85 (d,4H), 7.18 (d, 4H), 7.31 (d, 4H), 7.45 (d, 2H), 7.95 (d, 4H).

¹H NMR of 162 (400 MHz, CD₃OD) δ 3.8 (s, 6H), 6.4 (m, 4H), 6.95 (d, 4H),7.38 (d, 4H), 7.5 (d, 4H), 7.6 (d, 2H), 7.95 (d, 4H). MS ESI (neg ion)for [M−H]⁻: 621 (calculated 622).

General Method for the Synthesis of Compounds (A11)

By allowing a compound of formula (A10)-1 prepared as above to reactwith a sulfonyl chloride (R₂SO₂Cl), and subsequently by oxidizingintermediate 163 and by allowing intermediate 164 to react with athioamide (R₃C(S)NH2) wherein R₁, R₂ and R₃ are defined as above infonmula (A11).

The first step in this sequence of reactions may be carried out in asolvent such as tetrahydrofuran (THF), dichloromethane (CH₂Cl₂), in thepresence of a base (e.g. 4,4-dimethylaminopyridine, triethylamine,triisopropylaminc) and a sulfonyl chloride (e.g. tosyl chloride, mesylchloride), at temperatures ranging from −20° C. to 23° C., for 1 to 60hours. The second step in this sequence of reactions may be carried outin a solvent such as dichloromethant (CH₂Cl₂), in the presence of anoxidizing reagent (e.g. tetrapropylammonium perruthenate (VII) (TPAP))and activated 4 Å molecular sieves at temperatures ranging from 0° C. to23° C., for 1 to 60 hours. The third step in this sequence of reactionsmay be carried out in a solvent such as acetic acid, toluene, dioxane attemperatures ranging from 0° C. to 120° C., for 1 to 60 hours.

EXAMPLES

To 50 mg of diol 22 in 1 mL of dichloromethane was added Tosyl chloride(42.5 mg), 4,4-dimethylaminopyridine (6 mg), triethylamine (95 μl), andthe reaction was stirred for 12 hours at 23° C. The volatiles wereremoved in vacuo and the crude mixture (containing 165, thebis-tosylated compound and the corresponding epoxide) was separated byflash chromatography (ethyl acetate-hexane eluent) to give a mixture of165 and the corresponding bis-tosylated compound (27 mg total).

The mixture consisting of 165 and the corresponding bis-tosylatedcompound was oxidized as described above for compound 24. The crudemixture was purified by flash chromatograpby (ethyl acetae-hexaneeluent) to give 3.9 mg of 166 and 16 mg of the correspondingbis-tosylate. ¹H NMR of 166 (400 MHz, d₆-acetone) δ 1.5 (d, 18H), 2.4(s, 3H), 6.4 (d, 1H), 6.5 (d, 1H), 6.95(s, 1H), 7.35 (d, 2H), 7.42 (d,2H), 7.5 (d, 1H), 7.6 (m, 3H), 7.7 (dd, 4H), 8 (d, 2H).

To 3.9 mg of 166 was added 3 mg of para-methoxythiobenzamnide and 0.5 mlof toluene and the reaction was heated at 65° C. for 12 hours. Thesolvent was removed in vacuo and the crude mixture was purified by flashchromatography (ethyl actat-hexane eluent) to give 1.8 mg of 167. ¹H NMRof 167 (400 MHz, CDCl₃) δ 1.5 (d, 18H), 3.8 (s, 3H), 6.3 (dd, 2H), 6.9(d, 2H), 7.35 (d, 2H), 7.4 (m, 4H), 7.55 (m, 4H), 7.9 (d, 2H).

¹H NMR of 168 (400 MHz, CD₃OD) δ 3.8 (s, 3H), 6.45 (dd, 2H), 7.0 (d,2H), 7.4 (d, 2H), 7.5-7.7 (m, 8H), 7.9 (d, 2H).

BIOLOGICAL PROTOCOLS PTP-1B Gene Cloning and Protein Purification

The following procedure was conducted for recombinant production andpurification of protein tyrosine phosphatase PTP-1B, for use as asubstrate in PTPase inhibition assays.

A. Production of a PTP-1B cDNA

A human placental cDNA library was synthesized in a 50 ul reactioncontaining 1 ug human placental poly(A)⁺ mRNA (Clontech, Palo Alto,Calif.), 4 ul random hexamer primers, 8 ul of 10 mM dNTPs (Pharmacia,Piscataway N.J.), 1 ul (200 U/ul) Moloney murine leukemia virus reversetranscriptase (Gibco-BRL, Canada), 0.5 ul (26 U/ul) RNAsin (Promega,Madison Wis.), and 12 ul 5× buffer (Gibco-BRL). The synthesis reactionwas incubated at 37° C. for one hour and then heat inactivated at 95° C.for five minutes.

A PTP-1B cDNA was amplified, using polymerase chain reaction (PCR), fromthe cDNAs synthesized as described above. More particularly, based onthe published sequence of PTB-1B, two PCR primers were synthesized toamplify a portion of the PTB-1 B coding sequence known to encode a 321amnino acid fragment containing the PTP-1B catalytic domain and havingPTPase activity. See Hoppe et al., Eur. J. Biochem., 223:1069-77 (1994);Barford, D., et al., J. Molec. Biol., 239:726-730 (1994); Chernoff etal., Proc. Natl. Acad. Sci. USA, 87:2735-2739 (1990); Charbonneau et al.Proc. Natl. Acad. Sci. USA, 86:5252-5256 (1989). The primers had thefollowing respective sequences:

PTP-1B-A(5′) (SEQ ID NO: 1)

5 ′ CGCACTGGATCCTCATGGAGATGGAAAAGG 3′

PTP-1B-B(3′) (SEQ ID NO: 2)

5 ′ CTCCCTGAATTCCTAATTGTGTGGCTCCAGG 3′

The first primer, which hybridizes to the non-coding strand, correspondsto the 5′ portion of the PTP-1B coding sequence and encodes a BamH Irestriction site upstream of the initiation codon, to facilitatecloning. The second primer, which hybridizes to the coding strand,corresponds to the 3′ portion of the PTB-1B fragment of intest, andencodes a stop codon and an EcoR I restriction site downstream from thestop codon.

A 100 μl PCR reaction mixture containing approx. 1 ug of the humanplacental cDNA library, 0.2 mM of each dNTP, 30 uM of each primer, 1×Amplitaq DNA polymerase buffer (Perkin-Elmer, Norwalk Conn.), and 5units Amplitaq DNA polymerase (Perkin-Elmer) was denatured at 94° C. for5 minutes and then subjected to 25 cycles of amplification asfollows: 1) 94° C. denaturation for 1 minute; 2) 55° C. annealing for 1minute; and 3) 72° C. primer extension for 1 minute.

The PCR reaction product (992 bp) was digested with BamH I and EcoR I(New England Biolabs, Beverly Mass.) to yield a 975 bp product encodingthe 321 amino acid PTP-1B protein fragment, and having “sticky ends” tofacilitate cloning.

B. Production of a PTP-1B Expression Vector.

The 975 bp PTP-1B partial cDNA was purified by agarose gelelectrophoresis and ligated into a BamH I/EcoR I-digested pGEX-3Xplasmnid vector (Pharmacia, Piscataway, N.J.). The pGEX vector isdesigned to produce a fusion of glutathione-S-transferase (GST) to aprotein encoded by another DNA fragment inserted into the vector'scloning site. Complete sequencing of the insert of the resultantplasmid, designated pGEX-3X-PTP-1B, confirmed the identity of the PTP-1BcDNA, and insertion in the proper orientation and reading frame.

C. Expression and Purification of GST/PTB-1B Fusion Protein.

E. coli strain DH5α (Gibco-BRL) was transformed with plasmidpGEX-3X-PTP-1B following the supplier's transformation protocol andgrown at 37° C. with vigorous shaking in Luria-Bertani brothsupplemented with 100 ug/ml ampicillin. When the cultures reached anO.D.₆₀₀ of 0.7-1, production of the GST/PTP-1B fusion protein wasinduced with 0.1 mM IPTG (Isopropyl b-D-Thiogalactoside). After 3additional hours of culturing at 37° C., the bacteria were pelleted bycentrifugation.

The bacterial pellet was resuspended in 10× (w/v) lysis bufferconsisting of 12.5 mM HEPES, 2 mM EDTA, pH 7.0, 15 mM b-mercaptoethanol(bME) and 1 mM PMSF. The lysate was sonicated (on ice) until slightclearing was observed (approx. three min.) and then centrifuged at10,000 revolutions per minute (RPM) for 10 min. The supernatant wasdiluted 1:4 with buffer A (25 mM HEPES, pH 7.0, and 15 mM bME).

Primary purification was achieved using a 5 ml Hi-Trap pre-packed Qcolumn (Pharmacia). After loading the diluted supernatant onto thecolumn, the column was washed with 10 bed volumes of buffer A. TheGST/PTB-1B fusion protein was then eluted using a linear gradient ofBuffer A and Buffer B (buffer A+1M NaCl). Eluted fractions containingprotein were identified by SDS-PAGE and Coomassie Blue staining(Pharmacia PhastSystem), and fractions containing PTP-1B activity wereidentified using the PTP-1B activity assay described below. Elution ofthe fusion protein occurred at about 30% Buffer B.

Fractions contining TPase activity wrre pooled, diluted 1:4 with NETbuffer (20 mM Tris, pH 8.8, 100 mM NaCl, 1 mM EDTA and 15 mM bME), andloaded onto a 10 mnl GST-Sepharose 4B column (Pharmacia). After loading,the column was washed first with 3 bed volumes of NET buffer+1% NP40(Sigma Chemical Co., St Louis, Mo.), then with NET buffer until O.D. at280 nm was basal. The GST/PTP-1B fusion protein was eluted from thecolumn using 10 mM glutathione in 33 mM Tris, pH 8.0. Elution ofproteins was monitored at O.D.₂₈₀ and fractions were assayed foractivity and run on SDS-PAGE as described above. PTP-1B fusion proteineluted after approx. 4-5 minutes (flow rate 1 ml/min.).

The GST/PTP-1B-containing fractions from the GST-Sepharose 4Bpurification were pooled, concentrated into a final storage buffer (0.2MNaCl, 25 mM HEPES, 1 mM EDTA, and 5 mM DTT, pH 7.0) using a 1 ml Hi-TrapQ column (pre-packed, Pharmacia), and stored at −80° C. (finalconcentration of 0.52 mg/ml). The foregoing procedure yieldedapproximately 5 mg of PTP-1B fusion protein per 500 ml of culturedcells, purified to substantial homogeneity as assessed by SDS-PAGE.

Assay of PTP-LB Activity

PTP-1B enzymatic activity of samples was assayed in microtiter plates asfollows.

The protein concentration of the PTP-1 B enzyme preparation wasdetermined using the Bio-Rad Protein Assay kit (Bio-Rad, HerculesCalif.). An aliquot from each sample was taken and diluted to 2 mgprotein/mil using activity assay buffer (100 mM Sodium Acetate, pH 6.0,1 mM EDTA, 0.1% TX-100 (International Biotechnologies, Inc.) and 15 mMbME) to form a PTP-1B stock solution.

A 100 ul reaction mixture was prepared containing 10 ul of the PTP-1Bstock solution, 10 ul of 9 mM p-nitrophenylphosphate ((pNPP), SigmaChemical Co., St. Louis Mo.), and 80 ul of activity assay buffer (100 mMsodium acetate, pH 6.0, 1 mM EDTA, 0.1% Triton X-100, 15 mM bME).Reactions were mixed gently and incubated at 37° C. for 60 minutes.Enzymatic cleavage of phosphate from pNPP (a tyrosine phosphate analog)is marked by a colorimetric change in this substrate. See, e.g., Imbertet al., Biochem J., 297:163-173 (1994); Ghosh and Miller, Biochem.Biophys. Res. Comm., 194:36-44 (1993); Zanke et al., Eur. J. Immunol.,22:235-39 (1992).

Reactions were stopped by addition of 10 ul of a 0.5M NaOH/50% EtOHsolution. To determine the enzymatic activity, absorbance readings ofthe reactions were measured at 405 nm using a Molecular DevicesThermomax Plate Reader (Menlo Park Calif.).

CD45 Gene Cloning and Protein Purification

The following procedure was conducted for recombinant production andpurification of protein tyrosine phosphatase CD45, for use as asubstrate in PTPase inhibition assays.

A. Production of a CD45 cDNA, and production of a CD45 expressionvector.

A human cDNA library was synthesized from RNA isolated from the humanJurkat cell line, as described above for PTP-1B

CD45 cDNA was amplified, using polymerase chain reaction (PCR), from thecDNAs synthesized above. Two PCR primers were synthesized to amplify thecoding sequence of CD45. The primers had the following respectivesequences:

CD45 (5′) (SEQ ID NO: 3)

5′CTACATCCCGGGATGTCCTGCAATTTAGATG 3′

CD45 (3′) (SEQ ID NO: 4)

5′CATTTATGTCCCGGGCTATGAACCTTGAT 3′

The first primer corresponds to the 5′ portion of the CD45 codingsequence and encodes a Sma I restriction site upstream of the initiationcodon, to facilitate cloning. The second primer corresponds to the 3′portion of the CD45 sequnce, and encodes a stop codon and a Sma Irestriction site downstream firm the stop codon.

The PCR reaction product (2127 bp) was digested with Sma I (New EnglandBiolabs, Beverly Mass.) to yield a 2110 bp product. The pET24C plasmidvector (Novagen, Inc., Madison Wis.) was digested with the BamH Irestriction enzye, and the “sticky” ends were filled using T4 DNApolymerase according to the manufacturer's instructions (New EnglandBiolabs, Beverly Mass.); the resulting plasmid DNA was ligated to SmaI-digested CD45 PCR product. The pET24C vector is designed to producehigh levels of the protein encoded by cDNA inserted into the vector'scloning site (CD45), in bacterial hosts. Complete sequencing of theinsert of the resultant plasmid, designated pET24C-CD45, confirmed theidentity of the CD45cDNA, and insertion in the proper orientation andreading frame.

C. Expression and Purification of CD45 protein.

E. coli strain DH5α (Gibco-BRL) was transformed with pET24C-CD45following the supplier's transformation protocol, plated ontoLuria-Bertani agar plates supplemented with 30 ug/ml kanamycin and grownovernight at 37° C. A single bacterial colony was trnnsferred into 50mls Luria-Bertani broth supplemented with 30 ug/ml kanamycin and grownovernight with vigorous shaking. This overnight culture was split intotwo equal parts, and added to 2L Luria-Bertani broth supplemented with50 ug/ml kanamycin. When the cultures reached an O.D.₆₀₀ of 1,production of the recombinant CD45 protein was induced with 0.1 mM IPTG(Isopropyl b-D-Thiogalactoside). After 5 additional hours of culturingat 37° C., the bacteria were pelleted by centrifugation.

The bacterial pellet (approximately 5 grams) was resuspended in 10×(w/v)lysis buffer consisting of 12.5 mM HEPES, 2 mM EDTA, pH 7.0, 15 mM bMEand 1 mM PMSF. The lysate was sonicated (on ice) until slight clearingwas observed (approx. three min.) and then centrifuged at 10,000revolutions per minute (RPM) for 10 min. The supernatant was filteredthrough 1 mm Wattman filter paper, and 9.7 grams (i.e., 194 grams/L) ofanmnonium sulfate were added to the solution on ice to precipitatesoluble proteins. After a 1 hour incubation on ice, the lysate was spunat 10,000 RPM for 30 min. at 4 C; supernatant was removed, and anadditional 7.6 grams (i.e., 151 grans/L) of ammonium sulfate were added.The resulting pellet was resuspended in 3 mls of buffer B (33 mMimidazole-HCl pH 8.0, 2 mM EDTA, 10 mM bME, 0.002% PMSF) and stored onice. After another 1 hour incubation on ice, the spin supernatant withammonium sulfate was spun again at 10,000 RPM for 30 mins at 4 C. Theresulting pellet from the second centrifugation was resuspended in 2 mlsof buffer B. The two pellet solutions were pooled and dialyzed overnightagainst buffer B.

Secondary purification was achieved using a Mono-Q column. (Pharmacia).After loading the diluted supernatant onto the column, the column waswashed with 10 bed volumes of buffer B The recombinant CD45 protein wasthen eluted using a linear gradient of Buffer B and Buffer C (bufferB+1M NaCl). Eluted fractions containing protein were identified bySDS-PAGE and Coomassie Blue staining (Pharmacia PhastSystem), andfractions containing CD45 activity were identified using the CD45activity assay described below.

The CD45-containing fractions from the MonoQ column purification werepooled and stored at 4 C.

Assay of CD45 Activity

CD45enzymatic activity of samples was assayed in microliter plates asfollows.

A 100 ul reaction mixture was prepared containing 10 ul of the CD45stock solution, 10 ul of 9.3 mM p-nitrophenylphosphate ((pNPP), SigmaChemical Co., St. Louis Mo.), and 80 ul of activity assay buffer (100 mMsodium acetate, pH 6.0, 1 mM EDTA, 0.1% Triton X-100, 15 mM bME).Reactions were mixed gently and incubated at 37° C. for 60 minutes.Reactions were stopped by addition of 10 ul of a 0.5M NaOH/50% EtOHsolution. To determine the enzymatic activity, absorbance readings ofthe reactions were measured at 405 nm using a Molecular DevicesThermomax Plate Reader (Menlo Park Calif.).

In vitro PTPase Inhibition Assay

The ability of the compounds of the present invention, such as thecinnamic acid derivative compounds of Example 2, to inhibit the PTPaseactivity of PTP-1B, CD45, PTP-1C, and PTPα was determined usingmodifications of the PTP-1B and CD45 activity assays described inExamples 3 and 4.

First, 0.001 mmol of the cinnamic acid derivative (or other PTPaseinhibitor compound) was dissolved in 100 ul of DMSO to create a 10 mMstock solution. The 10 mM stock solution was used to add varyingconcentrations (100 uM, 33 uM, 10 uM, 3 uM, 1 uM, 0.3 uM, 0.1 uM, 0.03uM, 0.01 uM or 0.003 uM) of the inhibitor compound to a series ofotherwise identical PTPase activity assay reactions (100 ul final volumein microtiter wells). Thus, each 100 ul reaction contained 10 ul PTPaseenzyme stock solution (final phosphatase concentration of approximately20 ng/well), 70 ul activity assay buffer, 10 ul pNPP stock solution(final pNPP concentration of 0.9 mM for PTP-1B assay, 0.93 mM for CD45assay, 0.5 mM for PTPα assay, and 8 mM for PTP-1C assay), and 10 ul ofthe diluted inhibitor compound in DMSO. Assay buffers contained: forCD45 and PTP-1B assays, 100 mM sodium acetate at pH 6.0, 1 mM EDTA, 0.1%Triton X-100, and 15 mM bME; for PTP-1C assays, 100 mM sodium acetate atpH 5.5, 0.1% BSA, and 15 mM bME; for PTPα assays, 100 mM sodium acetateat pH 5.25, 0.1% BSA, and 15 mM bME. Purified phosphatase was added tothe reaction mixtures to begin the reactions; the reactions wereincubated at 37° C. for 60 min. (for PTP-1B and CD45 assays) or at 27 Cfor 60 min. (for PTP-1C and PTPα assays), stopped, and colorimetricallyanalyzed as described above. As positive and negative controls,reactions were performed containing 10 ul DMSO with no inhibitorcompound or containing the known PTPase inhibitors vanadate (finalconcentration 0.5 mM; for PTP-1B and CD45 assays) or ammnonium molybdate(final concentration 1 mM; for PTP-1C and PTPα assays) substituted forthe inhibitor compound of the invention.

The concentration of inhibitor compound required to inhibit 50% of thePTPase activity (IC50) was detemnined as follows. First, absorbancereadings from the negative control reactions were treated as a baselineand subtracted from the absorbance readings of the experimentalreactions. Then, for each reaction, a percent inhibition was calculatedusing the following formula:

100×[1−(O.D.₄₀₅reaction/O.D.₄₀₅DMSO)]

For each inhibitor compound tested, an IC50 concentration was calculatedfrom a best-fit computer aasis of the calculated percent inhibition forthe various dilutions of the compound.

Inhibitor compounds having an IC50 less than 10 uM (and optimally lessthan 5 uM) for a particular PTPase were scored as highly effectiveinhibitors of that PTPase enzyme, and are preferred inhibitors of thepresent invention.

As it will be apparent to those persons skilled in the art, theforegoing biological data is not absolute and will vary according tomany factors such as assay conditions and the like.

TABLE 8 % inhibition % inhibition % inhibition of PTP1B of PTPα of PTP1CCompound at 1 μM at 100 μM at 100 μM 36 52 0 42 37 85 63 59 38 93 71 6339 82 47 53 40 88 82 62 41 39 20 17 42 84 92 88 43 76 82 79 44 79 87 8645 85 85 84 46 75 73 61 47 68 48 63 48 69 3 33 49 37 0 35 50 50 37 25

TABLE 9 IC50 values (in μM) against PTP1B and CD45 for given compoundsPTP1B CD45 9 0.37 3.9 13 31 —* 23 0.27 —* 25 0.89 —* 27 0.5 —* 29 0.8 —*32 1.8 —* 54 0.072 0.73 55 0.1 0.56 56 0.135 0.94 57 0.25 1.0 58 0.250.97 59 0.25 0.35 60 0.29 1.0 61 0.97 0.955 62 1.5 0.985 63 1.7 2.4 643.0 6.4 65 1.3 1.4 66 1.7 2.5 67 1.0 1.25 68 0.3 0.865 69 0.41 1.9 700.42 1.9 71 0.43 0.53 72 0.52 5.5 73 0.62 2.8 74 0.64 4.2 75 0.68 3.4 760.68 0.93 77 0.78 7.5 78 0.79 1.15 79 4.8 8.2 80 10 20 81 26 19 82 11.912.8 83 1.3 1.5 84 1.2 2.7 85 1.5 1.8 86 1.8 7.1 87 1.0 1.1 88 2.65 7.889 13.7 >100 90 0.86 1.12 91 25.9 >100 94 0.7 7 95 2 6 96 0.4 2.4 97 610 98 6 10 99 1.5 7.4 100 26 >100 133 —* 134 3.4 20 136 0.7 8 140 1.2 20144 3 —* 146 5.9 —* 148 9 —* 152 0.85 1.2 153 2.65 1.91 154 3.83 2.45155 1 1.3 156 1.7 1.3 157 5.5 1.5 160 0.98 1.52 162 1.8 —* 168 3 —* —*:data not available

TABLE 10 % inhibition % inhibition of PTP1B of CD45 Compound at 1 μM at1 μM 103 44% 14% 104 24% 14% 105 61% 18% 106 45% 21% 107 25% 51% 108 30%62% 109 —* 14% 110 —* 22% 111 —* 18% 112 —* 16% 113 —* 61% —*: data notavailable

TABLE 11 % inhibition of PTP1B Compound at 1 μM 116 41% 117 67% 118 56%119 71% 120 67% 121 73% 122 87% 123 85% 124 83% 125 93% 126 59% 127 79%128 80%

The compounds of the present invention have asymmetric centers and mayoccur as racemates, racemic mixtures, and as individual enantiomers ordiastereoisomers, with all isomeric forms being included in the presentinvention as well as mixtures thereof.

Pharmaceutically acceptable salts of the compounds of Formula (A1) thru(A11) where a basic or acidic group is present in the structure, arealso included within the scope of this invention. When an acidicsubstituent is present, such as —COOH, there can be formed the ammonium,sodium, potassium, calcium salt, and the like, for use as the dosageform. When a basic group is present, such as amino or a basic heteroarylradical, such as pyridyl, an acidic salt, such as hydrochloride,hydrobromide, acetate, maleate, pamoate, methanesulfonate,p-toluenesulfonate, and the like, can be used as the dosage form.

Also, in the case of the —COOH being present, pharmaceuticallyacceptable esters can be employed, e.g., methyl, tert-butyl,pivaloyloxymethyl, and the like, and those esters known in the art formodifying solubility or hydrolysis characteristics for use as sustainedrelease or prodrug formulations.

In addition, some of the compounds of the instant invention may formsolvates with water or common organic solvents. Such solvates areencompassed within the scope of the invention.

The term “therapeutically effective amount” shall mean that amount ofdrug or pharmaceutical agent that will elicit the biological or medicalresponse of a tissue, system, animal, or human that is being sought by aresearcher, veterinarian, medical doctor or other clinician. Generally,a daily dose of about 0.5 mg/Kg to 100 mg/Kg body weight in divideddoses is suggested to treat PTPase related diseases. Such dosage has tobe individualized by the clinician.

The present invention also has the objective of providing suitabletopical, oral and parenteral pharmaceutical formulations for use in thenovel methods of treatment of the present invention. The compounds ofthe present invention may be administered orally as tablets, aqueous oroily suspensions, lozenges, troches, powders, granules, emulsions,capsules, syrups or elixirs. The composition for oral use may containone or more agents selected from the group of sweetening agents,flavouring agents, colouring agents and preserving agents in order toproduce pharmaceutically elegant and palatable preparations. The tabletscontain the acting ingredient in admixture with non-toxicpharmaceutically accepble excipients which are suitable for themanufacture of tablets. These excipients may be, for example, (1) inertdiluents , such as calcium carbonate, lactose, calcium phosphate orsodium phosphate; (2) granulating and disintegrating agents, such ascorn starch or alginic acid; (3) binding agents, such as starch, gelatinor acacia; and (4) lubricating agents, such as magnesium stearate,stearic acid or talc. These tablets may be uncoated or coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. Coating may also beperformed using techniques described in the U.S. Pat. Nos. 4,256,108;4,160,452; and 4,265,874 to form osmotic therapeutic tablets for controlrelease.

Formulations for oral use may be in the form of hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin. They may alsobe in the form of soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, such as peanut oil, liquid paraffinor olive oil.

Aqueous suspensions normally contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspension. Suchexpicients may be:

(1) suspending agent such as sodium carboxymethyl cellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia;

(2) dispersing or wetting agents which may be (a) naturally occurringphosphatide such as lecithin; (b) a condensation product of an alkyleneoxide with a fatty acid, for example, polyoxyethylene stearate; (c) acondensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethylenoxycetanol; (d) a condensationproduct of ethylene oxide with a it, partial ester derived from a fattyacid and hexitol such as polyoxyethylene sorbitol monooleate, or (e) acondensation product of ethylene oxide with a partial ester derived fromfatty acids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to known methods using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition.sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

Compounds of Formula (A1) thru (A11) may also be administered in theform of suppositories for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperature butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials are cocoa butter and polyethyleneglycols.

The compounds of the present invention may also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the compounds of Formula (A1) thru (A11) are employed.

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What is claimed is:
 1. A compound with the structure depicted in Formula(A9):

wherein at least one of R₁ and R₂ substituents has the structuredepicted in Formula (B)

wherein (i) R′ and R″ are independently selected from the groupconsisting of hydrogen, halo, cyano, nitro, trihalomethyl, C₁₋₁₁alkyl,substituted or unsubstituted arylC₁₋₁₁alkyl wherein the arylsubstituents are independently selected from the group consisting ofhydrogen, halo, nitro, cyano, trihalomethyl, hydroxypyronyl, C₁₋₁₁alkyl,arylC₁₋₁₁alkyl, C₀₋₁₁alkyloxyC₀₋₁₁alkyl, arylC₀₋₁₁alkyloxyC₀₋₁₁alkyl,C₀₋₁₁alkylthioC₀₋₁₁alkyl, arylC₀₋₁₁alkylthioC₀₋₁₁alkyl,C₀₋₁₁alkylamioC₀₋₁₁alkyl, arylC₀₋₁₁alkylaminoC₀₋₁₁alkyl,di(arylC₁₋₁₁alkyl)aminoC₀₋₁₁alkyl, C₁₋₁₁alkylcarbonylC₀₋₁₁alkyl,aryC₁₋₁₁alkylcarbonylC₀₋₁₁alkyl, C₀₋₁₁alkylcarboxyC₀₋₁₁alkyl,arylC₁₋₁₁alkylcarboxyC₀₋₁₁alkyl, C₁₋₁₁alkylcarbonylaminoC₀₋₁₁alkyl,arylC₁₋₁₁alkylcarbonylaminoC₀₋₁₁alkyl, —C₀₋₁₁alkylCOOR₄,—C₀₋₁₁alkylCONR₅R₆ wherein R₄, R₅ and R₆ are independently selected fromhydrogen, C₁-C₁₁alkyl, arylC₀-C₁₁alkyl, or R₅ and R₆ are taken togetherwith the nitrogen to which they are attached forming a ring systemcontaining 3 to 8 carbon atoms with at least one C₁-C₁₁alkyl,arylC₀-C₁₁alkyl substituent. (ii) R′″ is selected from the groupconsisting of (a) hydrogen, (b) C₁₋₁₁alkyl, substituted C₁₋₁₁alkylwherein the substituents are independently selected from halo, cyano,nitro, trihalomethyl, carbamoyl, tetrahydrofuryl, pyrrolidinyl,piperidinyl, morpholinyl, piperazinyl, hydroxypyronyl, C₀₋₁₁alkyloxy,arylC₀₋₁₁alkyloxy, C₀₋₁₁alkylthio, arylC₀₋₁₁alkylthio, C₀₋₁₁alkylamino,arylC₀₋₁₁alkylamino, di(arylC₀₋₁₁alkyl)amino, C₁₋₁₁alkylcarbonyl,arylC₁₋₁₁alkylcarbonyl, C₁₋₁₁alkylcarboxy, arylC₁₋₁₁alkylcarboxy,C₁₋₁₁alkylcarbonylamino, arylC₁₋₁₁alkylcarbonylamino,—C₀₋₁₁alkylCOOR₇,—C₀₋₁₁alkyCONR₈R₉ wherein R₇, R₈ and R₉ areindependently selected from hydrogen, C₁-C₁₁alkyl, arylC₀₋₁₁alkyl, or R₈and R₉ are taken together with the nitrogen to which they are attachedforming a ring system containing 3 to 8 carbon atoms with at least oneC₁-C₁₁alkyl, arylC₀-C₁₁ alkyl substituent, (c) mono-, di- andtri-substituted arylC₀-C₁₁ alkyl wherein the aryl substituents aredefined as above for R′ and R″, (iii) X is a mono-, di- ortrisubstituted aryl wherein the aryl substituents are defined as abovefor R′ and R″, and aryl is selected from phenyl, biphenyl, naphthyl,dihydronaphthyl, tetrahydronaphthyl, indenyl, indanyl, azulenyl,anthryl, phenanthryl, fluorenyl, pyrenyl, thienyl, benzothienyl,isobenzothienyl, 2,3-dihydrobenzothienyl, furyl, pyranyl, benzofuranyl,isobenzofuiranyl, 2,3-dihydrobenzofrranyl, pyrrolyl, indolyl,isoindolyl, indolizinyl, indazolyl, imidazolyl, benzimidazolyl, pyridyl,pyrazinyl, pyradazinyl, pyrimidinyl, triazinyl, quinolyl, isoquinolyl,4H-quinolizinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, chromanyl, benzodioxolyl, piperonyl,purinyl, hydroxypronyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl,isothiazolyl, benzthiazolyl, oxazolyl, isoxazolyl, benzoxazolyl,oxadiazolyl, or thiadiazolyl, and wherein the remaining of R₁ and R₂ isindependently selected from the group consisting of: (i) hydrogen; (ii)C₁₋₁₁alkyl, substituted C₁₋₁₁alkyl wherein the alkyl substituents aredefined as above, (iii) arylC₀₋₁₁alkyl, (iv) mono-, di- andtri-substituted arylC₀-C₁₁alkyl wherein the aryl substituents aredefined as above, and wherein m is an integer between 0 and 3 and eachR₃ is independently selected from the group consisting of hydrogen,halo, nitro, cyano, trihalomethyl, hydroxypyronyl, C₁₋₁₁alkyl,C₀₋₁₁alkyloxyC₀₋₁₁alkyl, arylC₀₋₁₁alkyloxyC₀₋₁₁alkyl,C₀₋₁₁alkylthioC₀₋₁₁alkyl, arylC₀₋₁₁alkylthioC₀₋₁₁alkyl,C₀₋₁₁alkylaminoC₀₋₁₁alkyl, arylC₀₋₁₁alkylaminoC₀₋₁₁alkyl,di(arylC₁₋₁₁alkyl)aminoC₀₋₁₁alkyl, C₀₋₁₁alkylcarbonylC₀₋₁₁alkyl,C₁₋₁₁alkylcarboxyC₀₋₁₁alkyl, C₁₋₁₁alkylcarbonylaminoC₀₋₁₁alkyl,arylC₁₋₁₁alkylcarbonylC₀₋₁₁alkyl, arylC₁₋₁₁alkylcarboxyC₀₋₁₁alkyl,arylC₀₋₁₁alkylcarbonylaminoC₀₋₁₁alyl, —CH═CHCOOR₁₀, —CH═CHCONR₁₁R₁₂,—C₀₋₁₁alkylCOOR₁₃, —C₀₋₁₁alkylCONR₁₄R₁₅ wherein R₁₀ thru R₁₅ areindependently selected from hydrogen, C₁-C₁₁ alkyl, arylC₀-C₁₁alkyl, orR₁₁ and R₁₂ are taken together with the nitrogen to which they areattached forming a ring system containing 3 to 8 carbon atoms with atleast one C₁-C₁₁alkyl arylC₀-C₁₁alkyl substituent, or R₁₄ and R₁₅ aretaken together with the nitrogen to which they are attached forming aring system containing 3 to 8 carbon atoms with at least oneC₁-C₁₁alkyl, arylC₀-C₁₁alkyl substituent, or its pharmaceuticallyacceptable salts, or solvates thereof.
 2. A compound as defined in claim1 wherein aryl is selected from phenyl, naphthyl, biphenyl, thienyl,furyl, pyridl, or its pharmaceutically acceptable salts, or solvatesthereof.
 3. A compound as defined in claim 1 wherein aryl is phenyl, orits pharmaceutically acceptable salts, or solvates thereof.
 4. Acompound as defined in claim 1 wherein aryl is naphthyl, or itspharmaceutically acceptable salts, or solvates thereof.
 5. A compound asdefined in claim 1 wherein aryl is biphenyl, or its pharmaceuticallyacceptable salts, or solvates thereof.
 6. A compound as defined in claim1 wherein aryl is thienyl, or its pharmaceutically acceptable salts, orsolvates thereof.
 7. A compound as defined in claim 1 wherein aryl isfuryl, or its pharmaceutically acceptable salts, or solvates thereof. 8.A compound as defined in claim 1 wherein aryl is pyridyl, or itspharmaceutically acceptable salts, or solvates thereof.
 9. A compoundhaving the structure depicted in Formula (A9):

wherein R₁ or R₂ is selected from —COR₁₆, —COOR₁₇, —CONR₁₈R₁₉ whereinR₁₆ thru R₁₉ are independently selected from hydrogen, C₁-C₁₁alkyl,substituted C₁₋₁₁alkyl where the alkyl substituents are as definedbelow, unsubstituted or substituted arylC₀-C₁₁alkyl where the arylsubstituents are as defined below, or R₁₈ and R₁₉ are taken togetherwith the nitrogen to which they are attached forming a ring systemcontaining 3 to 8 carbon atoms with at least one C₁-C₁₁alkyl,arylC₀-C₁₁alkyl substituents, and wherein the remainder of R₁ or R₂ hasthe general structure depicted in Formula (B)

wherein (i) R′ and R″ are independently selected from the groupconsisting of hydrogen, halo, cyano, nitro, trihalomethyl, C₀₋₁₁alkyl,substituted or unsubstituted arylC₁₋₁₁alky1 wherein the arylsubstituents are independently selected from the group consisting ofhydrogen, halo, nitro, cyano, trihalomethyl, hydroxypyronyl, C₁₋₁₁alkyl,arylC₁₋₁₁alkyl, C₀₋₁₁akyloxyC₀₋₁₁alkyl, arylC₀₋₁₁alkyloxyC₀₋₁₁alkyl,C₀₋₁₁alkylthioC₀₋₁₁alkyl, arylC₀₋₁₁alkylthioC₀₋₁₁alkyl,C₀₋₁₁alkylaminoC₀₋₁₁alkyl, arylC₀₋₁₁alkylaminoC₀₋₁₁alkyl,di(arylC₁₋₁₁alkyl)aminoC₀₋₁₁alkyl, C₁₋₁₁alkylcarbonylC₀₋₁₁alkyl,arylC₁₋₁₁alkylcarbonylC₀₋₁₁alkyl, C₁₋₁₁alkylcarboxyC₀₋₁₁alkyl,arylC₁₋₁₁alkylcarboxyC₀₋₁₁alkyl, C₁₋₁₁alkylcarbonylaminoC₀₋₁₁alkyl,arylC₁₋₁₁alkylcarbonylaminoC₀₋₁₁alkyl, —C₀₋₁₁alkylCOOR₄,—C₀₋₁₁alkylCONR₅R₆ wherein R₄, R₅ and R₆ are independently selected fromhydrogen, C₁-C₁₁alkyl, arylC₀-C₁₁alkyl, or R₅ and R₆ are taken togetherwith the nitrogen to which they are attached forming a ring systemcontaining 3 to 8 carbon atoms with at least one C₁-C₁₁alkyl,arylC₀-C₁₁alkyl substituent, (ii) R′″ is selected from the groupconsisting of (a) hydrogen, (b) C₁₋₁₁alkyl, substituted C₁₋₁₁alkylwherein the substituents are independently selected from halo, cyano,nitro, trihalomethyl, carbamoyl, tetrahydrofuryl, pyrrolidinyl,piperidinyl, morpholinyl, piperazinyl, hydroxypyronyl, C₀₋₁₁alkyloxy,arylC₀₋₁₁alkyloxy, C₀₋₁₁alkylthio, arylC₀₋₁₁alkylthio, C₀₋₁₁alkylamino,arylC₀₋₁₁alkylamino, di(arylC₀₋₁₁alkyl)amino, C₁₋₁₁alkylcarbonyl,arylC₁₋₁₁alkylcarbonyl, C₁₋₁₁alkylcarboxy, arylC₁₋₁₁alkylcarboxy,C₁₋₁₁alkylcarbonylamino, arylC₁₋₁₁alkylcarbonylamino, —C₀₋₁₁alkylCOOR₇,—C₀₋₁₁alkylCONR₈R₉ wherein R₇, R₈ and R₉ are independently selected fromhydrogen, C₁-C₁₁alkyl, arylC₀₋₁₁alkyl, or R₈ and R₉ are taken togetherwith the nitrogen to which they are attached forming a ring systemcontaining 3 to 8 carbon atoms with at least one C₁-C₁₁alkyl,arylC₀-C₁₁alkyl substituent, (c) mono-, di- and tri-substitutedarylC₀-C₁₁alkyl wherein the aryl substituents are defined as above forR′ and R″, (iii) X is a mono-, di- or trisubstituted aryl wherein thearyl substituents are defined as above for R′ and R″, and aryl isselected from phenyl, biphenyl, naphthyl, dihydronaphthyl,tetrahydronaphthyl, indenyl, indanyl, azulenyl, anthryl, phenanthryl,fluorenyl, pyrenyl, thienyl, benzothienyl, isobenzothienyl,2,3-dihydrobenzothienyl, ffryl, pyranyl, benzofuranyl, isobenzofuranyl,2,3-dihydrobenzofuranyl, pyrrolyl, indolyl, isoindolyl, indolizinyl,indazolyl, imidazolyl, benzimidazolyl, pyridyl, pyrazinyl, pyradazinyl,pyrimidinyl, triazinyl, quinolyl, isoquinolyl, 4H-quinolizinyl,cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, chromanyl, benzodioxolyl, piperonyl,purinyl, hydroxypronyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl,isothiazolyl, benzthiazolyl, oxazolyl, isoxazolyl, benzoxazolyl,oxadiazolyl, or thiadiazolyl, and wherein R₂ is selected from the groupconsisting of: (i) hydrogen; (ii) C₁₋₁₁alkyl, substituted C₁₋₁₁alkylwherein the alkyl substituents are defined as above, (iii)arylC₀₋₁₁alkyl, (iv) mono-, di- and tri-substituted arylC₀₋₁₁alkylwherein the aryl substituents are defined as above, and wherein m is aninteger between 0 and 3 and each R₃ is independently selected from thegroup consisting of hydrogen, halo, nitro, cyano, trihalomethyl,hydroxypyronyl, C₁₋₁₁alkyl, C₀₋₁₁alkyloxyC₀₋₁₁alkyl,arylC₀₋₁₁alkyloxyC₀₋₁₁alkyl, C₀₋₁₁alkylthioC₀₋₁₁alkyl,arylC₀₋₁₁alkylthioC₀₋₁₁alkyl, C₀₋₁₁alkylaminoC₀₋₁₁alkyl,arylC₀₋₁₁alkylaminoC₀₋₁₁alkyl, di(arylC₀₋₁₁alkyl)aminoC₀₋₁₁alkyl,C₀₋₁₁alkylcarbonylC₀₋₁₁alkyl, C₁₋₁₁alkylcarboxyC₀₋₁₁alkyl,C₁₋₁₁alkylcarbonylaminoC₀₋₁₁alkyl, arylC₁₋₁₁alkylcarbonylC₀₋₁₁alkyl,arylC₁₋₁₁alkylcarboxyC₀₋₁₁alkyl, arylC₁₋₁₁alkylcarbonylaminoC₀₋₁₁alkyl,—CH═CHCOOR₁₀, —CH═CHCONR₁₁R₁₂, C₀₋₁₁alkylCOOR₁₃, —C₀₋₁₁alkylCONR₁₄R₁₅wherein Rio thru R, ₅ are independently selected from hydrogen,C₁-C₁₁alkyl, arylC₁₀-C₁₁alkyl, or R₁₁, and R₁₂ are taken together withthe nitrogen to which they are attached forming a ring system containing3 to 8 carbon atoms with at least one C₁-C₁₁alkyl, arylC₀-C₁₁alkylsubstituent, or R₁₄ and R₁₅ are taken together with the nitrogen towhich they are attached forming a ring system containing 3 to 8 carbonatoms with at least one C₁-C₁₁ alkyl, arylC₀-C₁₁alkyl substituent, orits pharmaceutically acceptable salts, or solvates thereof.
 10. Acompound as defined in claim 9 wherein aryl is selected from phenyl,naphthyl, biphenyl, thienyl, furyl, pyridyl, or its pharmaceuticallyacceptable salts, or solvates thereof.
 11. A compound as defined inclaim 9 wherein aryl is phenyl, or its phanraceutically acceptablesalts, or solvates thereof.
 12. A compound as defined in claim 9 whereinaryl is naphthyl, or its pharmaceutically acceptable salts, or solvatesthereof.
 13. A compound as defined in claim 9 wherein aryl is biphenyl,or its pharmaceutically acceptable salts, or solvates thereof.
 14. Acompound as defined in claim 9 wherein aryl is thienyl, or itspharmaceutically acceptable salts, or solvates thereof.
 15. A compoundas defined in claim 9 wherein aryl is furyl, or its pharmaceuticallyacceptable salts, or solvates thereof.
 16. A compound as defined inclaim 9 wherein aryl is pyridyl, or its pharmaceutically acceptablesalts, or solvates thereof.